6-substituted estradiol derivatives and methods of use

ABSTRACT

Disclosed are compounds of the formula: 
                         
wherein R 1 , R 2 , R 3  and R 4  are independently hydrogen, C 1 -C 6  alkyl, halo, a sulfate, a glucuronide, —OH, a bulky group, aryl, cycloalkyl, heteroaryl, heterocycloalkyl, —N(CH 2 ) n ; a phosphate group, and a phosphinate group; R 9  is hydrogen, halogen or alkyl; R 11  is selected from the group consisting of H, C 1 -C 6  alkyl, halogen, a sulfate, a glucoronide, —SO 2 NH 2 , —COOH, —CN, —CH 2 CN—, —NHCN—, —CHO, ═CHOCH 3 , —COO salt, —OSO 2 alkyl, —NH 2 , and —NHCO(CH 2 ) n ; R 12  is selected from the group consisting of H, a C 1 -C 6  alkyl, a sulfate, a glucoronide, a bulky group, aryl, cycloalkyl, heteroaryl and heterocycloalkyl; X is selected from the group consisting of C 1 -C 12  alkyl, C 2 -C 12  alkenyl, C 2 -C 12  alkynyl, halogen, a glucoronide, —NH 2 , —SO 2 NH 2 , —COOH, —CN, —CH 2 CN, —NHCN, —CHO, —COOsalt, —OSO 2 alkyl, —SH, —SCH 3 , —CH[(CH 2 ) n CH 3 ]COOCH 3 , —(CH 2 ) m COOCH 3 , —(CH 2 ) m —O—CH 3 , —(CH 2 ) m —O—(CH 2 ) n CH 3 , (CH 2 ) m —S—CH 3 , —(CH 2 ) m —S—(CH 2 ) n CH 3 , —(CH 2 ) m —NH—(CH 2 ) n CH 3 , —C 2 -C 8  alkenyl-O—(CH 2 ) n CH 3 , —C 2 -C 8  alkenyl-S—(CH 2 ) n CH 3 , —C 2 -C 8  alkenyl-N—(CH 2 ) n CH 3 , —C 2 -C 8  alkynyl-O—(CH 2 ) n CH 3 , —C 2 -C 8  alkynyl-S—(CH 2 ) n CH 3 , —C 2 -C 8  alkynyl-N—(CH 2 ) n CH 3 , —(CH 2 ) m —OH, —(CH 2 ) m —O—NH 2 , —(CH 2 ) m —S—NH 2 , —NH(CH 2 ) m CH 3 , —NH(CH 2 ) m OCH 3 , —NH(CH 2 ) m CHOH—COOH, —N(CH 3 ) 2 , —(CH 2 ) m (NH)CH 2 OH, —NHCOOH, —(CH 2 ) m NHCOOH, —NO 2 , —SCN, —SO 2 alkyl, —B(OH) 2 , —(CH 2 ) m N(CH 3 )—SO 2 —NH 3 , —(CH 2 ) m —NH—SO 2 —NH 2 , —NHC(═S) CH 3 , and —NHNH 2 ; and Y is selected from hydrogen, ═O, —OCO(R 6 ) and —OH; wherein m is an integer between 0-20, n is an integer between 0-8, the   symbol represents either a single or a double bond capable of forming a keto group at position 3 or 17; and the   symbol represents any type of bond regardless of the stereochemistry; and the respective enantiomers, other stereochemical isomers, hydrates, solvates, tautomers and pharmaceutically acceptable salts of said compounds. The compounds are useful in the treatment of various types of cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.12/132,857 filed on Jun. 4, 2008 now U.S. Pat. No. 8,168,621, which is acontinuation-in-part of U.S. application Ser. No. 11/947,645 filed onNov. 29, 2007, which claims the benefit of U.S. Provisional ApplicationNo. 60/867,980 filed Nov. 30, 2006. U.S. application Ser. No. 11/947,645also is continuation-in-part of U.S. application Ser. No. 11/541,987filed on Oct. 2, 2006 now U.S. Pat. No. 7,846,918, which claims thepriority benefit from U.S. Provisional Application No. 60/722,204 filedSep. 30, 2005. The teachings of all such applications are incorporatedherein by their entirety.

FIELD OF THE INVENTION

The present invention relates to compositions and methods of making andusing 6-substituted estradiol compounds and their pharmaceuticallyacceptable salts or prodrugs thereof as articulated and describedherein. The present invention also pertains to pharmaceuticalcompositions comprising such compounds, present either in vitro or invivo, for both diagnostic applications and also treatment ofproliferative conditions, such as cancer.

BACKGROUND OF THE INVENTION

Proliferative cell disorders such as tumors and primary malignant tumors{herein, cancer(s)} in particular are problematic given their tendencyto invade surrounding tissues and metastasize to distant organs in thebody. To date the most frequently used methods for treating neoplasia,especially solid tumor forms of neoplasia, include surgical procedures,radiation therapy, drug chemotherapies, and combinations of theforegoing.

With over million cases of cancer being diagnosed annually, and cancerclaiming more than half a million lives in the United States each year,there is increased need in new therapeutic modalities against suchcondition. Prostate, lung and colorectal remain the most common cancersamong men; while breast, colorectal and lung cancers are the most commoncancers among women.

In recent years, there have been significant gains in the management ofthese conditions. At least one of the success stories in the clinicalmanagement of a cancer is the early diagnosis and treatment options nowavailable for primary breast cancer. The other is employment ofeffective and nontoxic anti-estrogen agents that block the actions ofestrogen either at its receptor sites or at a point of its synthesis.

Obviously research on the function and activity of estrogen receptors,the structure and their function has been the subject of many recentinvestigations. Estrogen receptors belong to a large family ofstructurally related ligand-inducible transcription factors, includingsteroid receptors, thyroid/retinoid receptors, vitamin D receptors knownas nuclear receptors. While the true ligand for nuclear receptors havenot been described, there are distinct small molecules that are able tobind to such receptors and trigger a cellular response.

Estrogens and estrogen receptor modulators bind to estrogen receptors,classified into two types; α and β, to form discrete molecular complexesthat exert pleiotropic tissue-specific effects by modulating theexpression of target genes. The ligand-bound estrogen receptor acts as akey transcription factor in various molecular pathways, and modulationof ER expression levels is important in determining cellular growthpotential.

While both these types of receptors bind to estrogen, as well as otheragonists and antagonists, the two receptors have distinctly differentlocalization concentration within the body. Aside from some structuraldifferences between the α and β types, when complexes with estrogen, thetwo were shown to signal in opposite way, with estrogen activatingtranscription in the presence of Estrogen Receptor α (ERα) andinhibiting transcription in the presence of Estrogen Receptor β (ERβ).

Tamoxifen is primarily one of the first selective estrogen receptormodulators that have become first-line therapy for hormonal treatment ofbreast cancer, both for adjuvant treatment and for therapy of metastaticdisease. Tamoxifen is a competitive inhibitor of estradiol binding tothe estrogen receptor inhibiting its estrogen binding to the estrogenbinding element on DNA. It has been suggested that Tamoxifen's bindingto the estrogen receptors significantly alters the structuralconfiguration of the estrogen receptors, rendering the binding sitesdysfunctional towards any endogenous estrogen. Such structuraldeformation of the receptor could explain the profound side effectprofile associated with the use of Tamoxifen.

At least another shortcoming of Tamoxifen is its ineffectiveness againstnon-estrogen-dependent tumors and lower efficacy in pre-menopausalwomen. Additionally, Tamoxifen undergoes an isomerization underphysiological conditions from a therapeutically useful antiestrogeniccompound to an estrogenic isomer which can stimulate the growth ofestrogen-dependent tumor cells, providing an undesired clinical outcome,particularly among patients suffering from estrogen dependent tumors.

U.S. Pat. No. 4,732,904 discloses other type of estrogen receptorantagonists conventionally known as hydrazone compounds. It is thoughtthat these antiestrogenic hydrazone compounds do not undergoisomerization to estrogenic compounds under physiological conditions andthe estrogenic side effects observed for Tamoxifen are therefore absent.These hydrazone compounds have been proposed as alternative treatmentsfor estrogen-dependent breast cancers. Among these, the substitutedbenzophenone nitrophenyl hydrazones, such as4,4′-dihydroxybenzophenone-2,4-dinitrophenylhydrazone are described tobe superior.

The complex of the receptor and the antiestrogen such as hydrazone basedcompounds or Tamoxifen may then bind to nuclear chromatin in an atypicalmanner for a longer time than the normal hormone receptor complex.Antiestrogens may also be able to deplete the cytoplasm of freereceptor. Either or both of these effects could severely impair thecontinued growth of an estrogen-dependent tumor.

There has also been an increased interest in the use of aromataseinhibitors to block specifically the local production of estrogens thatmay contribute substantially to hormone responsive disease such asbreast cancer. Aromatase (CYP19) is described as the principal enzymethat converts androgens to estrogens both in pre- and postmenopausalwomen. Estrogen deprivation through aromatase inhibition is described asan effective and selective treatment for some postmenopausal patientswith hormone-dependent breast cancer.

Exemestane (which is sold as Aromasin, is chemically described as6-methylenandrosta-1,4-diene-3,17-dione) and acts as an irreversible,steroidal aromatase inactivator. It is believed to act as a falsesubstrate for the aromatase enzyme, and processed to an intermediatethat binds irreversibly to the active site of the enzyme causing itsinactivation. U.S. Pat. No. 4,808,616, and U.S. Pat. No. 4,904,650, theteachings of which are incorporated herein in their entirety, disclose6-alkylidenandrosta-1,4-diene-3,17-dione derivatives, such asexemestane, and methods of making them. U.S. Pat. No. 4,876,045discloses a method of preparing 6-methylene derivatives ofandrosta-1,4-diene-3,17-diones. U.S. Pat. No. 4,990,635 discloses aprocess for making 6-methylene derivatives ofandrosta-1,4-diene-3,17-diones.

The preparation of intermediates that may be useful in preparingexemestane is disclosed in U.S. Pat. No. 3,274,176. In German patent DD258820, 6-hydroxymethyl-androsta-1,4-diene-3,17-dione is prepared fromandrosta-1,4-diene-3,17-dione via1,3-dipyrrolidinoandrosta-3,5-dien-17-one.

Co-pending international application no. PCT/US2005/001248 filed Jan.14, 2005 (PCT Publication Number WO 2005/070951), incorporated herein byreference in its entirety, also describes the preparation ofintermediates that are useful in preparing exemestane. The structure ofExemestane is shown below.

Schneider et. al, in “Course of the reaction of steroidal 3,5-dienamineswith formaldehyde”, Helvetica Chimica Acta (1973), 56(7), 2396-2404,discloses the following compounds:

where the

symbol represents a double bond, it means a keto group and no R₆ ispresent; and where the

symbol represents a single bond R₆ is hydrogen (i.e. an alcohol group).Unlike the compounds of the present invention, Schneider's compounds donot embrace estradiol, testostrone or dihydrotestostrone variations.

A tri-hydroxyl substituted derivative of estranes is disclosed in U.S.Pat. No. 3,377,363 to Tadanier et. al, and the 3 hydroxy substituent onthe aromatic ring of the present compounds is not disclosed.

U.S. Pat. No. 5,914,324 to De Funari et. al, discloses 6-hydroxy and oxyandrostane derivatives for hypertension and heart failure. U.S. Pat. No.6,384,250 to Gobbini, et al., discloses the hydroxyl and ketonesubstituents at the 6 position in the preparation of (E,Z)3-(2-aminoethoxyimino)-androstane-6,17-dione. These compounds weredirected towards the treatment of heart failure. The effects of alkylhydroxyl substitution at the 6 position is not disclosed.

Tanenbaum, et. al, “Crystallographic comparison of the estrogen andprogesterone receptor's ligand binding domains”, Proc. Natl. Acad. Sci.USA, Biochemistry, Vol. 95, pp 5998-6003, discloses the mechanism of ERreceptors and notes that estradiol containing an aromatic ring with a3-hydroxy substituent binds well with the ER ligand binding region. Itis disclosed that a flat aromatic group without the 19 methylsubstituent is favored.

U.S. Pat. No. 5,892,069 to D'Amato describes estradiol derivatives thatinhibit tubulin polymerization during cell mitosis. Given the above, aneed still exists to identify new and effective agents for treatingcancer.

Another point of concern in the field is the eventual conversion of someestrogen-dependent cancers, i.e. breast cancer, to estrogen-independenttypes. This may be accounted for by a natural loss of differentiation bythe tumor cells. Estrogen-dependent cancer cells have often beenobserved to eventually lose their ability to produce estrogen-bindingprotein receptors and degenerate into much more aggressiveestrogen-independent life-threatening cancers. Indeed, the use ofantiestrogens to treat estrogen-dependent tumors may lead to the clonalselection of estrogen-independent tumor cells and therefore may promotethe conversion of an estrogen-dependent cancer to anon-estrogen-dependent cancer.

Cancers of other organs, such as lung and colon, may not concernestrogen-binding protein receptors and thus are considered independentof estrogens for cell replication. Such estrogen-independent tumors arenot as susceptible to the antiestrogenic properties of drugs such asTamoxifen, aromatase inhibitors. Thus other chemotherapeutic agents mustbe used to treat such tumors. Many compounds have been documented to beeffective to varying degrees against estrogen-independent tumors.

These compounds are reviewed in many references and typicallyadministered in combination regimen chemotherapy causing substantialside effect to the patients. The underlying principle of using generalcytotoxic agents in chemotherapy is based upon the observation thatmalignant tumor cells replicate at a higher rate than normal body cellsand are therefore correspondingly more susceptible to these compounds.Similarly, normal tissues that proliferate rapidly (for example, bonemarrow and intestinal epithelium) are subject to substantial damage onceexposed to these potent cytotoxic drugs, and such toxicity often limitsutility.

On the other hand, slow growing tumors with a small growth fraction, forexample carcinomas of the colon or lung, are often unresponsive tocytotoxic drugs. Aside from the treatment of estrogen-dependent andestrogen-independent tumors, many of the cytotoxic drugs are currentlybeing used for other proliferative diseases with rapidly growing cellsinvolved non-cancerous or non-malignant hyperproliferative conditions.

Also the increasing importance of effective therapeutic management ofviral diseases such as AIDS, herpes, various types of hepatitis andbacterial infections, especially among immune suppressed patients, callsfor alternative modes of therapy with favorable side effect profile.

Accordingly, there is not only a need for new and improved cancerchemotherapeutics that can be used to treat both estrogen-dependent andestrogen-independent tumors with minimal risk of systemic toxicitychallenging the quality of life for such fragile population of patients,but also for therapeutic remedies that target non-canceroushyperproliferative conditions which can benefit from effective doses ofestradiol derivatives. The hyperproliferative cells can be normal,rapidly growing cells or abnormal cells and can include tissue havingrapidly growing endogenous cells or their abnormal subpopulation, orother tissues generally exogenous to the patient.

None of the teachings of prior art provide for a therapeutic estradiolderivative with favorable side effect profile that can be used for thesetypes of conditions.

SUMMARY OF THE INVENTION

In light of the foregoing, the present invention is directed towardschemotherapeutic compounds, compositions and methods for their use andpreparation, thereby overcoming various deficiencies and shortcomings ofthe prior art, including those outlined above. Accordingly, it is oneobject of the present invention to provide compounds useful in thetreatment of estrogen-dependent conditions and tumors which provide abetter patient tolerance, prognosis and compliance.

Another object of the present invention is to provide compounds andmethods for the treatment of estrogen-independent tumors with compoundshaving substantially less side effects than those currently available tothe patients.

Yet another objective of the present invention is to provide forcompounds and alternative modes of treatment of tissues afflicted withhyperproliferative conditions, including viral and bacterial infections.

The present invention includes any one of the following sets ofcompounds represented in Formulas I-IV. One aspect of the presentinvention pertains to a compound of Formula I and II.

wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ are independently selectedfrom the group consisting of hydrogen, C₁ to C₆ alkyl or substitutedalkyl, halogen, sulfate, and glucuronide moieties; and the

symbol represents either a single or a double bond and when the

symbol is a double bond and forms a keto group at position 3 or 17, thenno R₇ or R₆ is present, respectively; the symbol --- represent thepresence or absence of a bond at position 10; and the

symbol represents any type of bond regardless of the stereochemistry.The compounds also embrace the enantiomers, other stereochemicalisomers, hydrates, solvates, tautomers and pharmaceutically acceptablesalts thereof.

In another aspect of the invention, compounds having Formula III aredescribed, wherein R₅ is as described above.

wherein R₅ is as described above.

Examples of compounds of Formulas (I-III) are shown below:

Another aspect of the present invention pertains to amine derivatives ofthe compounds of Formulas (I)-(III). In at least this aspect of theinvention, amine moieties are placed in suitable positions on themolecular core to improve physical and clinical properties. Formula (IV)represents a general core structure for the present invention. Formula(IV) depicts compounds having the structure:

wherein R₁, R₂, R₃ and R₄ are independently hydrogen, C₁-C₆ alkyl, halo,a sulfate, a glucuronide, —OH, a bulky group, aryl, cycloalkyl,heteroaryl, heterocycloalkyl, —N(CH₂)_(n); a phosphate group, and aphosphinate group; R₁₁ is selected from the group consisting of H, C₁-C₆alkyl, halogen, a sulfate, a glucoronide, —SO₂NH₂, —COOH, —CN, —CH₂CN—,—NHCN—, —CHO, ═CHOCH₃, —COO salt, —OSO₂alkyl, —NH₂, and —NHCO(CH₂)_(n);R₁₂ is selected from the group consisting of H, a C₁-C₆ alkyl, asulfate, a glucoronide, a bulky group, aryl, cycloalkyl, heteroaryl andheterocycloalkyl; X is selected from the group consisting of C₁-C₁₂alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, halogen, a glucoronide, —NH₂,—SO₂NH₂, —COOH, —CN, —CH₂CN, —NHCN, —CHO, —COOsalt, —OSO₂alkyl, —SH,—SCH₃, —CH[(CH₂)_(n)CH₃]COOCH₃, —(CH₂)_(m)COOCH₃, —(CH₂)_(m)—O—CH₃,—(CH₂)_(m)—O—(CH₂)_(n)CH₃, (CH₂)_(m)—S—CH₃, —(CH₂)_(m)—S—(CH₂)_(n)CH₃,—(CH₂)_(m)—NH—(CH₂)_(n)CH₃, —C₂-C₈ alkenyl-O—(CH₂)_(n)CH₃, —C₂-C₈alkenyl-S—(CH₂)_(n)CH₃, —C₂-C₈ alkenyl-N—(CH₂)_(n)CH₃, —C₂-C₈alkynyl-O—(CH₂)_(n)CH₃, —C₂-C₈ alkynyl-S—(CH₂)_(n)CH₃, —C₂-C₈alkynyl-N—(CH₂)_(n)CH₃, —(CH₂)_(m)—OH, —(CH₂)_(m)—NH₂, —(CH₂)_(m)—O—NH₂,—(CH₂)_(m)—S—NH₂, —NH(CH₂)_(m)CH₃, —NH(CH₂)_(m)OCH₃,—NH(CH₂)_(m)CHOH—COOH, —N(CH₃)₂, —(CH₂)_(m)(NH)CH₂OH, —NHCOOH,—(CH₂)_(m)NHCOOH, —NO₂, —SCN, —SO₂alkyl, —B(OH)₂,—(CH₂)_(m)N(CH₃)—SO₂—NH₃, —(CH₂)_(m)—NH—SO₂—NH₂, —NHC(═S)CH₃, and—NHNH₂; and Y is selected from hydrogen, ═O, —OCO(R₆) and —OH; wherein mis an integer between 0-20, n is an integer between 0-8, the

symbol represents either a single or a double bond capable of forming aketo group at position 3 or 17; and the

symbol represents any type of bond regardless of the stereochemistry;and the respective enantiomers, other stereochemical isomers, hydrates,solvates, tautomers and pharmaceutically acceptable salts of saidcompounds.

Specific examples of compounds of Formula (IV) are shown below:

In another aspect of the invention, methods of inhibiting growth ofcancer cells comprising providing to a patient a prodrug of FormulasI-IV are described. As such, the compounds of the present invention maybe used for administration in a mammalian subject in the form of a drug,prodrug or active metabolite. However, it is envisioned that suchcompounds are most effective when incorporated into nanoparticles,liposomes or polymeric matrix systems or other delivery systems whichare capable of being directly delivered to a solid mass or be targetedto tissues of interest via suitable targeting agents.

The present invention also relates to a method of therapeuticallytreating cancer in a mammalian subject (e.g., a human patient). In thisaspect of the invention, methods are provided for inhibiting tumor orcancerous cell growth within the mammalian subject. In such a method,the cells are exposed to or contacted with a compound of formulas I-IVas described herein or pharmaceutically acceptable enantiomers, otherstereochemical isomers, hydrates, solvates, tautomers, or salts thereof,as shown herein. In a specific, non-limiting embodiment of the methodsof the present invention, a compound of Formula (I)-(IV) is used totherapeutically treat an identified cancer state as described herein. Inanother specific non-limiting embodiment of the methods of the presentinvention, a composition comprising a compound of Formula (I)-(IV) isused to therapeutically treat an identified cancer state as describedherein.

At least another aspect of the invention concerns delivery systems thatallows conversion of suitable analogues which can be converted to aspecified active compound in vivo after it is administered to thepatient for exerting its therapeutic activity.

The compounds of the present invention may be used to treat any tumorwhich may be either directly or indirectly effected by hormonal and/orestrogen-related activity, including but not in any way limited to solidtumors associated with breast, pancreatic, lung, colon, prostate,ovarian cancers, as well as brain, liver, spleen, kidney, lymph node,small intestine, blood cells, bone, stomach, endometrium, testicular,ovary, central nervous system, skin, head and neck, esophagus, or bonemarrow cancer; as well as hematological cancers, such as leukemia, acutepromyelocytic leukemia, lymphoma, multiple myeloma, myelodysplasia,myeloproliferative disease, or refractory anemia.

The compounds of the present invention may also be used incombination-based therapeutic cancer treatments in a mammalian subject.Such methods may comprise administration of a compound of Formulas(I)-(IV) in combination with other adjunct cancer therapies, such aschemotherapy, radiotherapy, gene therapy, hormone therapy and othercancer therapies known in the art.

Any of the compounds of the present invention may be contemplated foradministration to the mammalian subject in the form of a drug, prodrugor even active metabolite. In the methods of treatment of the presentinvention, the term “administering” shall encompass the treatment of thevarious conditions described with the compound specifically disclosed orwith a compound which may not be specifically disclosed, but whichconverts to the specified compound in vivo after administration to thepatient and exhibits therapeutic activity.

Other objects, features, benefits and advantages of the presentinvention will be apparent from this summary and the followingdescriptions of certain embodiments, and will be readily apparent tothose skilled in the art having knowledge of various chemotherapeuticcompounds, methods and/or modes of operation.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs and shall be understood to have themeanings described below. All publications and patents referred toherein are incorporated by reference in their entirety. Unless otherwisespecified, a reference to a particular compound includes all suchisomeric forms, including racemic and other mixtures thereof. Unlessotherwise specified, a reference to a particular compound also includesionic, salt, solvate (e.g., hydrate), protected forms, prodrugs, andother stereoisomers thereof, for example, as discussed herein.

It may be convenient or desirable to prepare, purify, and/or handle acorresponding salt of the active compound, for example, apharmaceutically-acceptable salt. Examples of pharmaceuticallyacceptable salts are discussed in Berge et al., 1977, “PharmaceuticallyAcceptable Salts,” J. Pharm. Sci., Vol. 66, pp. 1-19, and discussedherein.

Anti-proliferative compounds of the present invention have applicationin the treatment of cancer, and so the present invention furtherprovides anti-cancer agents. The term “anti-cancer agent” as usedherein, pertains to a compound which treats, delays progression,prolongs relapse period of, and controls symptoms of a cancer (i.e., acompound which is useful in the treatment of a cancer). The anti-cancereffect may arise through one or more mechanisms, including but notlimited to, the regulation of cell proliferation, the inhibition ofangiogenesis (the formation of new blood vessels), the inhibition ofmetastasis (the spread of a tumor from its origin), the inhibition ofinvasion (the spread of tumor cells into neighboring normal structures),or the promotion of apoptosis (programmed cell death), or tumor necrosisor tumor autophagy or any combinations thereof.

The invention further provides active compounds for use in a method oftreatment of the human or animal body by therapy. Such a method maycomprise administering to such a subject a therapeutically-effectiveamount of an active compound, preferably in the form of a pharmaceuticalcomposition as discussed further herein.

The term “estrogen” as used herein encompass steroid like hormones thatare naturally made and is able to cross the cell membrane to exert itsactivity inside the cell by binding to the estrogen receptors. Exampleof such compounds include but are not limited to estradiols, estrols,and esterenes.

The term “treatment,” or “therapy” as used herein in the context oftreating a condition, pertains generally to treatment and therapy of amammalian subject, whether of a human or a non-human animal (e.g., inveterinary applications), in which some desired therapeutic effect isachieved, for example, the inhibition of the progress of the condition,and includes a reduction in the rate of progress, a halt in the rate ofprogress, amelioration of the condition, and/or cure of the condition.Treatment as a prophylactic measure is also included. Treatment includescombination treatments and therapies, in which two or more treatments ortherapies are combined, for example, sequentially or simultaneously.Examples of treatments and therapies include, but are not limited to,chemotherapy (the administration of active agents, including, e.g.,drugs, antibodies (e.g., as in immunotherapy), prodrugs (e.g., employingprotecting groups including phosphoric acid derivatives and phosphinatesat suitable positions such as position 3 or 17, other compounds used forphotodynamic therapy, GDEPT, ADEPT, etc.); surgery; radiation therapy;and gene therapy.

The term “stereochemical isomer” as used herein, refers to isomers thatdiffer from each other only in the way the atoms are oriented in space.The two stereoisomers particularly of importance in the instantinvention are enantiomers and diastereomers depending on whether or notthe two isomers are mirror images of each other. In the preferredembodiment, the claimed formulations comprise such compounds thatisolated, resolved and are “substantially free of other isomers.”

The term “therapeutically-effective amount,” as used herein, pertains tothat amount of an active compound, or a material, composition or dosageform comprising an active compound, which is effective for producingsome desired therapeutic effect, commensurate with a reasonablebenefit/risk ratio.

The term “patient” refers to animals, including mammals, preferablyhumans.

The term “region of a patient” refers to a particular area or portion ofthe patient afflicted with a proliferative disorder, cancer or tumor andin some instances to regions throughout the entire patient. Exemplary ofsuch regions are the pulmonary region, the gastrointestinal region, thebreast region, the renal region as well as other bodily regions,tissues, lymphocytes, receptors, organs and the like, including thevasculature and circulatory system, and cancerous tissue. “Region of apatient” includes, for example, regions to be treated with the disclosedcompounds and compositions. The “region of a patient” is preferablyinternal, although it may be external.

The term “tissue” refers generally to specialized cells which mayperform a particular function. The term “tissue” may refer to anindividual cell or a plurality or aggregate of cells, for example,membranes, blood or organs. The term “tissue” also includes reference toan abnormal cell or a plurality of abnormal cells. Exemplary tissuesinclude breast tissue, including breast cells, membranous tissues,including endothelium and epithelium, laminae, connective tissue,including interstitial tissue, and tumors.

By “alkyl” in the present invention is meant a straight or branchedchain alkyl radical having 1-20, and preferably from 1-12, carbon atoms.Examples include but are not limited to methyl, ethyl, propyl,isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, 2-pentyl, isopentyl,neopentyl, hexyl, 2-hexyl, 3-hexyl, and 3-methylpentyl. Each alkyl groupmay be optionally substituted with one, two or three substituents suchas, for example, a halo, cycloalkyl, aryl, alkenyl or alkoxy group andthe like.

By “aryl” is meant an aromatic carbocylic radical having a single ring(e.g. phenyl), multiple rings (e.g. biphenyl) or multiple fused rings inwhich at least one is aromatic (e.g. 1,2,3,4-tetrahydronaphthyl). Thearyl group can also be optionally mono-, di-, or trisubstituted with,for example, halo, alkyl, alkenyl, cycloalkyl or alkoxy and the like.

By “heteroaryl” is meant one or multiple fused aromatic ring systems of5-, 6- or 7-membered rings containing at least one and up to fourheteroatoms selected from nitrogen, oxygen or sulfur. Examples includebut are not limited to furanyl, thienyl, pyridinyl, pyrimidinyl,benzimidazolyl and benzoxazolyl. The heteroaryl group can also beoptionally mono-, di-, or trisubstituted with, for example, halo, alkyl,alkenyl, cycloalkyl or alkoxy and the like.

By “cycloalkyl” is meant a carbocylic radical having a single ring (e.g.cyclohexyl), multiple rings (e.g. bicyclohexyl) or multiple fused rings(e.g.). The cycloalkyl group can optionally contain from 1 to 4heteroatoms. In addition, the cycloalkyl group may have one or moredouble bonds. The cycloalkyl group can also be optionally mono-, di-, ortrisubstituted with, for example, halo, alkyl, alkenyl, aryl or alkoxyand the like.

By “alkoxy” is meant an oxy-containing radical having an alkyl portion.Examples include, but are not limited to, methoxy, ethoxy, propoxy,butoxy and tert-butoxy. The alkoxy group can also be optionally mono-,di-, or trisubstituted with, for example, halo, aryl, cycloalkyl oralkoxy and the like.

By “alkenyl” is meant a straight or branched hydrocarbon radical havingfrom 2 to 20, and preferably from 2-6, carbon atoms and from one tothree double bonds and includes, for example, ethenyl, propenyl,1-but-3-enyl, 1-pent-3-enyl, 1-hex-5-enyl. The alkenyl group can also beoptionally mono-, di-, or trisubstituted with, for example, halo, aryl,cycloalkyl or alkoxy and the like.

“Halo” or “halogen” is a halogen radical of fluorine, chlorine, bromineor iodine.

By “glucuronide” is meant a glycoside radical of glucuronic acid.

The term “sulfate” refers to a radical having the general formula—OS(O)₂—OR′, wherein R′ is hydrogen, a metal or an alkyl group.

The term “phosphate” refers to a radical having the general formula—OP(O) (OR′)₂, wherein each R′ is independently hydrogen, a metal or analkyl group.

The term “phosphinate” refers to a radical having the general formula—OP(O) (R′)₂, wherein each R′ is independently hydrogen, a metal or analkyl group.

By “bulky group” is meant a substituent that produces steric hindranceabout the space to which it is attached, e.g. a t-butyl group.

The term “amino alkyl” as used herein refers to an alkyl group with anamino group on it, for example, H₂N—CH₂—, H₂N—CH₂CH₂—, Me₂NCH₂—, etc.,wherein the point of attachment is a carbon of the alkyl chain; and theterm “alkyl amino” as used herein refers to an amino group with an alkylgroup attached to the nitrogen atom, for example, CH₃NH—, EtNH—,iPr-NH—, etc., wherein the point of attachment is via the nitrogen atomof the amino group. All other terms wherein successive radicals areemployed will adhere to a similar rule.

The term “proliferative cell disorders” as used herein refers todisorders such as tumors, primary malignant tumors, and otherhyperproliferative conditions. The terms “primary malignant tumor(s)”and “cancer(s)” are used interchangeably.

Compounds

Among other things, the present invention relates to estradiolderivatives with specific modifications at position 6 of the B ring ofthe estradiol. At least one aspect of this invention is directed to suchcompounds having the general structure of Formula (IV) shown above.

In an embodiment of the present invention, preferred compounds have thegeneral structure shown in Formula (IVa) below:

wherein R₂, R₃, R₄, X and Y are as defined above for Formula (IV). Evenmore preferably, Y is selected from ═O and —OH; R₄ is selected fromhydrogen, halo and C₁-C₆ alkyl; R₂ is selected from hydrogen, —OH andhalo; R₃ is selected from hydrogen, halo and —OH; and X is selected fromC₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, —(CH₂)_(m)COOCH₃, —(CH₂)_(m)—O—CH₃,—(CH₂)_(m)—O—(CH₂)_(n)CH₃, (CH₂)_(m)—S—CH₃, —(CH₂)_(m)—S—(CH₂)_(n)CH₃,—(CH₂)_(m)—N—(CH₂)_(n)CH₃, —C₂-C₈ alkenyl-O—(CH₂)_(n)CH₃, —C₂-C₈alkenyl-S—(CH₂)_(n)CH₃, —C₂-C₈ alkenyl-N—(CH₂)_(n)CH₃, —C₂-C₈alkynyl-O—(CH₂)_(n)CH₃, —C₂-C₈ alkynyl-S—(CH₂)_(n)CH₃, —C₂-C₈alkynyl-N—(CH₂)_(n)CH₃, —(CH₂)_(m)—OH, —(CH₂)_(m)—O—NH₂,—(CH₂)_(m)—S—NH₂, —NH(CH₂)_(m)CH₃, —NH(CH₂)_(m)OCH₃,—NH(CH₂)_(m)CHOH—COOH, —(CH₂)_(m)(NH) CH₂OH, —(CH₂)_(m)NHCOOH,—(CH₂)_(m)N(CH₃)—SO₂—NH₃, and —(CH₂)_(m)—NH—SO₂—NH₂; m is an integerfrom 1-20; n is an integer from 0-8; and the

symbol represents either a single or a double bond. Yet even morepreferably, Y is (S)—OH; R₄ is selected from hydrogen or alkyl; R₂ ishydrogen; R₃ is hydrogen; and X is selected from C₁-C₁₂ alkyl, C₂-C₁₂alkenyl, —(CH₂)_(m)—O—CH₃, —(CH₂)_(m)—O—(CH₂)_(n)CH₃, (CH₂)_(m)—S—CH₃,and —(CH₂)_(m)—S—(CH₂)_(n)CH₃; m is an integer from 1-12; n is aninteger from 0-4; and the C-13 methyl is in the (S) configuration.

Yet another embodiment of the present invention is directed to achemotherapeutic compound of a Formulas (IVb):

wherein R₁ R₂, R₃, R₄ and X are as defined above for Formula (IV). Evenmore preferably, R₁ is selected from hydrogen, —OH and halo; R₄ isselected from hydrogen, halo and C₁-C₆ alkyl; R₂ is selected fromhydrogen and halo; R₃ is selected from hydrogen, halo and —OH; and X isselected from C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, —(CH₂)_(m)COOCH₃,—(CH₂)_(m)—O—CH₃, —(CH₂)_(m)—O—(CH₂)_(n)CH₃, (CH₂)_(m)—S—CH₃,—(CH₂)_(m)—S—(CH₂)_(n)CH₃, —(CH₂)_(m)—N—(CH₂)_(n)CH₃, —C₂-C₈alkenyl-O—(CH₂)_(n)CH₃, —C₂-C₈ alkenyl-S—(CH₂)_(n)CH₃, —C₂-C₈alkenyl-N—(CH₂)_(n)CH₃, —C₂-C₈ alkynyl-O—(CH₂)_(n)CH₃, —C₂-C₈alkynyl-S—(CH₂)_(n)CH₃, —C₂-C₈ alkynyl-N—(CH₂)_(n)CH₃, —(CH₂)_(m)—OH,—(CH₂)_(m)—O—NH₂, —(CH₂)_(m)—S—NH₂, —NH(CH₂)_(m)CH₃, NH(CH₂)_(m)OCH₃,—NH(CH₂)_(m)CHOH—COOH, —(CH₂)_(m)(NH) CH₂OH, —(CH₂)_(m)NHCOOH,—(CH₂)_(m)N(CH₃)—SO₂—NH₃, and —(CH₂)_(m)—NH—SO₂—NH₂; m is an integerfrom 1-20; and n is an integer from 0-8. Yet even more preferably, R₁ ishydrogen; R₄ is selected from hydrogen or alkyl; R₂ is hydrogen; R₃ ishydrogen; and X is selected from C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl,—(CH₂)_(m)—O—CH₃, —(CH₂)_(m)—O—(CH₂)_(n)CH₃, (CH₂)_(m)—S—CH₃, and—(CH₂)_(m)—S—(CH₂)_(n)CH₃; m is an integer from 1-12; n is an integerfrom 0-4; and both the C-13 methyl and C-17 hydroxyl are in the (S)configuration.

Still another embodiment of the invention, directed to a compound of aFormulas (IVc):

wherein R₁₁, R₂, R₃, R₄ and X are as defined above for Formula (IV).Even more preferably, R₁₁ is hydrogen or C₁-C₆ alkyl; R₄ is selectedfrom hydrogen, halo and C₁-C₆ alkyl; R₂ is selected from hydrogen andhalo; R₃ is selected from hydrogen, halo and —OH; and X is selected fromC₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, —(CH₂)_(m)COOCH₃, —(CH₂)_(m)—O—CH₃,—(CH₂)_(m)—O—(CH₂)_(n)CH₃, (CH₂)_(m)—S—CH₃, —(CH₂)_(m)—S—(CH₂)_(n)CH₃,—(CH₂)_(m)—N—(CH₂)_(n)CH₃, —C₂-C₈ alkenyl-O—(CH₂)_(n)CH₃, —C₂-C₈alkenyl-S—(CH₂)_(n)CH₃, —C₂-C₈ alkenyl-N—(CH₂)_(n)CH₃, —C₂-C₈alkynyl-O—(CH₂)_(n)CH₃, —C₂-C₈ alkynyl-S—(CH₂)_(n)CH₃, —C₂-C₈alkynyl-N—(CH₂)_(n)CH₃, —(CH₂)_(m)—OH, —(CH₂)_(m)—O—NH₂,—(CH₂)_(m)—S—NH₂, —NH(CH₂)_(m)CH₃, NH(CH₂)_(m)OCH₃,—NH(CH₂)_(m)CHOH—COOH, —(CH₂)_(m)(NH) CH₂OH, —(CH₂)_(m)NHCOOH,—(CH₂)_(m)N(CH₃)—SO₂—NH₃, and —(CH₂)_(m)—NH—SO₂—NH₂; m is an integerfrom 1-20; and n is an integer from 0-8. Yet even more preferably, R₁₁is hydrogen; R₄ is selected from hydrogen or alkyl; R₂ is hydrogen; R₃is hydrogen; and X is selected from C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl,—(CH₂)_(m)—O—CH₃, —(CH₂)_(m)—O—(CH₂)_(n)CH₃, (CH₂)_(m)—S—CH₃, and—(CH₂)_(m)—S—(CH₂)_(n)CH₃; m is an integer from 1-12; n is an integerfrom 0-4; and both the C-13 methyl and C-17 hydroxyl are in the (S)configuration.

Yet another embodiment of the present invention is directed to achemotherapeutic compound of a Formulas (IVd):

wherein R₁, R₂, and X are as defined above for Formula (IV). Even morepreferably, R₁ is selected from hydrogen, —OH and halo; R₂ is selectedfrom hydrogen and halo; and X is selected from C₁-C₁₂ alkyl, C₂-C₁₂alkenyl, —(CH₂)_(m)COOCH₃, —(CH₂)_(m)—O—CH₃, —(CH₂)_(m)—O—(CH₂)_(n)CH₃,(CH₂)_(m)—S—CH₃, —(CH₂)_(m)—S—(CH₂)_(n)CH₃, —(CH₂)_(m)—N—(CH₂)_(n)CH₃,—C₂-C₈ alkenyl-O—(CH₂)_(n)CH₃, —C₂-C₈ alkenyl-S—(CH₂)_(n)CH₃, —C₂-C₈alkenyl-N—(CH₂)_(n)CH₃, —C₂-C₈ alkynyl-O—(CH₂)_(n)CH₃, —C₂-C₈alkynyl-S—(CH₂)_(n)CH₃, —C₂-C₈ alkynyl-N—(CH₂)_(n)CH₃, —(CH₂)_(m)—OH,—(CH₂)_(m)—O—NH₂, —(CH₂)_(m)—S—NH₂, —NH(CH₂)_(m)CH₃, NH(CH₂)_(m)OCH₃,—NH(CH₂)_(m)CHOH—COOH, —(CH₂)_(m)(NH) CH₂OH, —(CH₂)_(m)NHCOOH,—(CH₂)_(m)N(CH₃)—SO₂—NH₃, and —(CH₂)_(m)—NH—SO₂—NH₂; X is selected fromC₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, —(CH₂)_(m)—O—CH₃,—(CH₂)_(m)—O—(CH₂)_(n)CH₃, (CH₂)_(m)—S—CH₃, and—(CH₂)_(m)—S—(CH₂)_(n)CH₃; m is an integer from 1-20; and n is aninteger from 0-8. Still even more preferably, R₁, R₂, R₃ and R₄ arehydrogen; m is an integer from 1-12; n is an integer from 0-4; and boththe C-13 methyl and C-17 hydroxyl are in the (S) configuration.

Yet another embodiment of the present invention is directed to achemotherapeutic compound of a Formulas (IVe):

wherein m, n, R₁, R₂, R₃ and R₄ are as defined above for Formula (IV),and Z is selected from —O—, —S— and —NH—. Even more preferably, m is1-12, n is 0-4, R₁ is selected from hydrogen, —OH and halo; R₄ isselected from hydrogen, halo and C₁-C₆ alkyl; R₂ is selected fromhydrogen and halo; R₃ is selected from hydrogen, halo and —OH; Z isselected from —O— and —S—; and both the C-13 methyl and C-17 hydroxylare in the (S) configuration.

Still another embodiment of the present invention is directed to achemotherapeutic compound of a Formulas (IVf):

wherein R₁, R₂, R₃, R₄ and X are as defined above for Formula (IV). Evenmore preferably, R₁ is selected from hydrogen, —OH and halo; R₄ isselected from hydrogen, halo and C₁-C₆ alkyl; R₂ is selected fromhydrogen and halo; R₃ is selected from hydrogen, halo and —OH; and X isselected from C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, —(CH₂)_(m)COOCH₃,—(CH₂)_(m)—O—CH₃, —(CH₂)_(m)—O—(CH₂)_(n)CH₃, (CH₂)_(m)—S—CH₃,—(CH₂)_(m)—S—(CH₂)_(n)CH₃, —(CH₂)_(m)—N—(CH₂)_(n)CH₃, —C₂-C₈alkenyl-O—(CH₂)_(n)CH₃, —C₂-C₈ alkenyl-S—(CH₂)_(n)CH₃, —C₂-C₈alkenyl-N—(CH₂)_(n)CH₃, —C₂-C₈ alkynyl-O—(CH₂)_(n)CH₃, —C₂-C₈alkynyl-S—(CH₂)_(n)CH₃, —C₂-C₈ alkynyl-N—(CH₂)_(n)CH₃, —(CH₂)_(m)—OH,—(CH₂)_(m)—O—NH₂, —(CH₂)_(m)—S—NH₂, —NH(CH₂)_(m)CH₃, NH(CH₂)_(m)OCH₃,—NH(CH₂)_(m)CHOH—COOH, —(CH₂)_(m)(NH) CH₂OH, —(CH₂)_(m)NHCOOH,—(CH₂)_(m)N(CH₃)—SO₂—NH₃, and —(CH₂)_(m)—NH—SO₂—NH₂; X is selected fromC₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, —(CH₂)_(m)—O—CH₃,—(CH₂)_(m)—O—(CH₂)_(n)CH₃, (CH₂)_(m)—S—CH₃, and—(CH₂)_(m)—S—(CH₂)_(n)CH₃; m is an integer from 1-20; and n is aninteger from 0-8. Still even more preferably, R₁, R₂, R₃ and R₄ arehydrogen; m is an integer from 1-12; and n is an integer from 0-4.

Embodiment compounds of the present invention can be used in apharmaceutical composition. Such a composition can comprise one or morecompounds selected from those discussed above, illustrated below orotherwise inferred herein, and combinations thereof. In certainembodiments, such a composition can comprise apharmaceutically-acceptable carrier component. Without limitation, sucha composition can comprise a racemic mixture of compounds. In certainembodiments, such a compound can be present as the S and R enantiomer,preferably their isolated and purified form which is substantially freeof other isomers, and R₅, or R₇ can be selected from H, C₁ to C₆ alkylor substituted alkyl, and a halogen.

The compounds of the present invention may have asymmetric centers andmay occur as a racemate, a racemic mixture or as individual and purifieddiastereomers or enantiomers such as (named via ChemDraw Ultra, Version11.0(3) or 12.0)(6S,8R,9S,13S,14S)-3-hydroxy-6-(methoxymethyl)-13-methyl-7,8,9,11,12,13,15,16-octahydro-6H-cyclopenta[a]phenanthren-17(14H)-one(compound 1);(6R,8R,9S,13S,14S)-3-hydroxy-6-(methoxymethyl)-13-methyl-7,8,9,11,12,13,15,16-octahydro-6H-cyclopenta[a]phenanthren-17(14H)-one(compound 2);(6S,8R,9S,13S,14S)-6-(methoxymethyl)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diol(compound 3);(6R,8R,9S,13S,14S)-6-(methoxymethyl)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diol(compound 4);(6S,8R,9S,10R,13S,14S)-17-hydroxy-6-(methoxymethyl)-10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one(compound 5);(6R,8R,9S,10R,13S,14S)-17-hydroxy-6-(methoxymethyl)-10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one(compound 6);(6S,8R,9S,13S,14S)-6-(hydroxymethyl)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diol(compound 7);(6R,8R,9S,13S,14S)-6-(hydroxymethyl)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diol(compound 8);(6R,8R,9S,10R,13S,14S)-6-(methoxymethyl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-3,17-diol(compound 9);(6R,8R,9S,13S,14S)-6-((aminooxy)methyl)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diol(compound 10);(6S,8R,9S,13S,14S)-6-((aminooxy)methyl)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diol(compound 11);(6R,8R,9S,13S,14S)-6-((aminooxy)methyl)-17-hydroxy-13-methyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one(compound 12);(6S,8R,9S,13S,14S)-6-((aminooxy)methyl)-17-hydroxy-13-methyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one(compound 13);(6R,8R,9S,13S,14S)-6-(((methoxymethyl)amino)methyl)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diol(compound 14);(6S,8R,9S,13S,14S)-6-(((methoxymethyl)amino)methyl)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diol(compound 15);1-((((6R,8R,9S,13S,14S)-3,17-dihydroxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-6-yl)methyl)amino)propan-2-one(compound 16);1-((((6S,8R,9S,13S,14S)-3,17-dihydroxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-6-yl)methyl)amino)propan-2-one(compound 17);(6R,8R,9S,13S,14S)-6-methoxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diol(compound 18);(6S,8R,9S,13S,14S)-6-(2-methoxyethyl)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diol(compound 19);(6R,8R,9S,13S,14S)-6-(4-methoxybutyl)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diol(compound 20);(6R,8R,9S,13S,14S)-6-(6-methoxyhexyl)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diol(compound 21);(6R,8R,9S,13S,14S)-6-(6-methoxyoctyl)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diol(compound 22);(6R,8R,9S,13S,14S)-3-hydroxy-6-(methoxymethyl)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-17-ylstearate (compound 23);(6R,8R,9S,10R,13S,14S)-6-(methoxymethyl)-10,13-dimethyl-7,8,9,10,11,12,13,14,15,16-decahydro-3H-cyclopenta[a]phenanthrene-3,17(6H)-dione(compound 24);(6S,8R,9S,10R,13S,14S)-6-(methoxymethyl)-10,13-dimethyl-7,8,9,10,11,12,13,14,15,16-decahydro-3H-cyclopenta[a]phenanthrene-3,17(6H)-dione(compound 25);(6R,8R,9S,10R,13S,14S)-6-(methoxymethyl)-10,13-dimethyl-4,5,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3H-cyclopenta[a]phenanthrene-3,17-diol(compound 26);(6S,8R,9S,10R,13S,14S)-6-(methoxymethyl)-10,13-dimethyl-4,5,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3H-cyclopenta[a]phenanthrene-3,17-diol(compound 27);(6S,8R,9S,13S,14S)-6-(methoxymethyl)-13-methyl-17-oxo-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-3-ylhydrogen sulfate (compound 28);(6R,8R,9S,13S,14S)-6-(methoxymethyl)-13-methyl-17-oxo-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-3-ylhydrogen sulfate (compound 29);(6R,8R,9S,13S,14S)-13-methyl-6-(4-propoxybutyl)-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diol(compound 30); and(6R,8R,9S,13S,14S)-13-methyl-6-(5-ethoxypentyl)-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diol(compound 31).

An embodiment of the present invention pertains to the preparation ofthe R or S enantiomers, and/or R or S diastereomers of 6 substitutedestradiols. Methods for the preparation (e.g., asymmetric synthesis) andseparation (e.g., fractional crystallization and chromatographic means)of such isomeric forms are either generally known in the art or arereadily obtained by adapting the methods taught herein. Suchmethodologies are, for example, described in the co-pending U.S.application U.S. Ser. No. 11/541,987, the teachings of which are hereinincorporated in its entirety.

Another embodiment of present invention pertains to a method forpreparing a 6-hydroxymethyl, 6-alkoxyalkyl, 6-alkylthioalkyl,6-aminomethoxy, 6-methylaminomethoxy, or 6-methoxyamine derivatives ofestradiol. Reaction schemes for preparing estradiol derivatives is givenbelow, Schemes 1-3. Such methods can comprise reaction of at-butyldimethylsilyl derivative of estradiol withLIDAKOR/THF/formaldehyde to obtain a 6-hydroxylated compound followed bysuch steps as: (i) hydrolysis to obtain 6-hydroxymethyl derivative ofestradiol; and/or (ii) treatment with dimethylsulfate followed byhydrolysis to obtain 6-methyloxymethyl derivative of estradiol. NDC-1088can be obtained by further oxidation of NDC-1033 at the C-17 hydroxylposition.

In an alternative approach, the compounds of the present invention canalso be prepared by a method comprising such steps as: (i) protecting anestrodial compound, (ii) acylating the protected estradiol compound atthe benzylic 6-position withLIDAKOR/Butyl-Lithium/Diisopropylamine/potassium tert-amylate, (iii)reducing the position 6 aldehyde with lithium aluminum hydride, (iv)deprotecting the protected regions of the estradiol compound. A reactionscheme for preparing estradiol derivatives is given below in Scheme 2.

The compounds of the present invention can be synthesized by thefollowing methods as depicted in the schemes below.

Various alkyloxyalkyl derivatives, in accordance with this invention,involve selection of alkylating agents. Such derivatives would beunderstood by those skilled in art made aware of this invention, and isavailable through synthetic procedures of the sort described herein.Accordingly, without limitation, various C₁ to C₆ alkyl and substitutedalkyl reagents can be used as described herein to prepare thecorresponding alkyloxyalkyl derivatives.

In another aspect of the invention, methods of making 6-aminoderivatives of the estradiol are disclosed in reaction schemes below.Accordingly, 6-methoxylated estradiols described in Schemes 1-2 areemployed and converted to their respective amino derivatives.

Methods of Use

The present invention relates to a method of treating cancer in amammalian subject (e.g., a human patient). In this aspect of theinvention, methods are provided for inhibiting tumor or cancerous cellgrowth. In such a method, the cells are exposed to or contacted with acompound of Formulas I-IV, including IVa-IVf, or pharmaceuticallyacceptable salts or hydrates thereof.

In at least another aspect of the present invention, effective doses ofcompounds having Formulas (I)-(IV), including IVa-IVf, are administeredto the patients in need of such therapy.

These methods may be used to treat any tumor which may be eitherdirectly or indirectly effected by hormonal and/or estrogen-relatedactivity, including but not in any way limited to solid tumorsassociated with breast, pancreatic, lung, colon, prostate, ovariancancers, as well as brain, liver, spleen, kidney, lymph node, smallintestine, blood cells, bone, stomach, endometrium, testicular, ovary,central nervous system, skin, head and neck, esophagus, or bone marrowcancer; as well as hematological cancers, such as leukemia, acutepromyelocytic leukemia, lymphoma, multiple myeloma, myelodysplasia,myeloproliferative disease, or refractory anemia.

Among other things, the invention offers a new mode of action fortreating estrogen dependent or independent tumors. Traditional approachemployed drugs once bound to the ERs modified the ERs configuration tothe extent that in effect rendered them destroyed. Accordingly,destruction of such bound ERs would cease transmission of all externaland internal signals essential for vitality of the cells, creating astop in cellular growth.

It is believed that the presently disclosed compounds are able to bindto number of receptors including the estrogen, testosterone and androgenreceptors. The inventor has unexpectedly observed that upon binding, thecompounds of the present invention are able to modulate the cellularfirst or second messenger signaling pathways and further potentiatetheir clinical effects through gene dependent or gene independentmechanisms, e.g. gene dependent estrogen activity has been welldescribed in the art and those of ordinary skill in the art are able toascertain the pathways involved inactivation of a estrogen dependentgene.

However, in the present invention, the claimed compounds are able tomodulate cellular activity at a level independent of the traditionalgene regulated mechanisms. In this aspect of the invention, thecompounds of the instant invention are capable of binding directly tomultiple steroid receptors at the plasma membrane and trigger internalcell mediated stress mechanisms involving the unfolded protein response(“UPR”) at the endoplasmic reticulum. The UPR stress responsesubsequently lead to growth inhibition, and cell death throughmodulation of stress response genes such as CHOP also known as GADD153,TRIB3, etc.

In addition, administration of the compounds of the present inventionfor treatment of various cancer states may comprise administration of acompound of formulas I-IV, including IVa-IVf, in combination with otheradjunct cancer therapies, such as chemotherapy, radiotherapy, genetherapy, hormone therapy and other cancer therapies known in the art.Combinations of the presently disclosed compounds with other anti-canceror chemotherapeutic agents are within the scope of the invention.Examples of such agents can be found in Cancer Principles and Practiceof Oncology by V. T. Devita and S. Hellman (editors), 6^(th) edition(Feb. 15, 2001), Lippincott Williams & Wilkins Publishers. A physician,veterinarian or clinician of ordinary skill in the art would be able todiscern which combinations of agents would be useful based on theparticular characteristics of the drugs and the cancer involved. Suchanti-cancer agents include the following: estrogen receptor modulators,androgen receptor modulators, retinoid receptor modulators, cytotoxicagents, anti-proliferative agents, prenyl-protein transferaseinhibitors, HMG-CoA reductase inhibitors, EHV protease inhibitors,reverse transcriptase inhibitors, aromatase inhibitors, and angiogenesisinhibitors.

Exemplified Compounds

In at least one aspect of the invention, the compounds of the presentinvention include those of table I below:

TABLE I

Spatial Substituents Configuration Entry R₅ R₆ R₇ C-6 C-8 C-9 C-13 C-14C-17  1 H H H S S S S S S  2 H H H S R R R R R  3 H H H S S S S S R  4 HH H S R R R R S Compound 7  H H H S R S S S S Compound 8  H H H R R S SS S  5 H — H S S S S S C═O  6 H — H S R R R R C═O  7 H H H R R R R R R 8 H H H R S S S S S  9 H H H R S S S S R 10 H H H R R R R R S 11 H — HR S S S S C═O 12 H — H R R R R R C═O 13 Me H H S S S S S S 14 Me H H S RR R R R Compound 3  Me H H S R S S S S 16 Me H H S R R R R S 17 Me — H SS S S S C═O 18 Me — H S R R R R C═O 19 Me H H R R R R R R Compound 4  MeH H R R S S S S 21 Me H H R S S S S R 22 Me H H R R R R R S 23 Me — H RS S S S C═O 24 Me — H R R R R R C═O Compound 1  H — H S R S S S C═OCompound 2  H — H R R S S S C═O 25 H H SO₃H S S S S S S 26 H H SO₃H S RR R R R 27 H H SO₃H S S S S S R 28 H H SO₃H S R R R R S 29 H — SO₃H S SS S S C═O 30 H — SO₃H S R R R R C═O 31 H H SO₃H R R R R R R 32 H H SO₃HR S S S S S 33 H H SO₃H R S S S S R 34 H H SO₃H R R R R R S 35 H — SO₃HR S S S S C═O 36 H — SO₃H R R R R R C═O 37 Me H SO₃H S S S S S S 38 Me HSO₃H S R R R R R 39 Me H SO₃H S S S S S R 40 Me H SO₃H S R R R R S 41 Me— SO₃H S S S S S C═O 42 Me — SO₃H S R R R R C═O 43 Me H SO₃H R R R R R R44 Me H SO₃H R S S S S S 45 Me H SO₃H R S S S S R 46 Me H SO₃H R R R R RS 47 Me — SO₃H R S S S S C═O 48 Me — SO₃H R R R R R C═O Compound 28 H —SO₃H S R S S S C═O Compound 29 H — SO₃H R R S S S C═O 49 H H glucuronideS S S S S S 50 H H glucuronide S R R R R R 51 H H glucuronide S S S S SR 52 H H glucuronide S R R R R S 53 H — glucuronide S S S S S C═O 54 H —glucuronide S R R R R C═O 55 H H glucuronide R R R R R R 56 H Hglucuronide R S S S S S 57 H H glucuronide R S S S S R 58 H Hglucuronide R R R R R S 59 H — glucuronide R S S S S C═O 60 H —glucuronide R R R R R C═O 61 Me H glucuronide S S S S S S 62 Me Hglucuronide S R R R R R 63 Me H glucuronide S S S S S R 64 Me Hglucuronide S R R R R S 65 Me — glucuronide S S S S S C═O 66 Me —glucuronide S R R R R C═O 67 Me H glucuronide R R R R R R 68 Me Hglucuronide R S S S S S 69 Me H glucuronide R S S S S R 70 Me Hglucuronide R R R R R S 71 Me — glucuronide R S S S S C═O 72 Me —glucuronide R R R R R C═O Compound 23 Me C(O)—(CH₂)₁₆CH₃ H R R S S S SCompound 10 NH₂ H H R R S S S S Compound 11 NH₂ H H S R S S S S R₁, R₂,R₃, R₄: independently H, C₁-C₆ alkyl, substituted alkyl, or halogen R₅,R₇: H, C₁-C₆ alkyl, substituted alkyl, sulfate, or glucuronide R₆: C₁-C₆alkyl, or substituted alkyl, sulfate, or glucuronide, when  

  is a single bond; not present, when  

  is a double bond

The preferred compounds in Table I include compounds 1, 2, 3, 4, 7, 8,10, 11, 23, 28 and 29. At least one aspect of the instant invention isdirected to these preferred compound, their method of use and making.

In at least another aspect of the invention, the compounds of thepresent invention are illustrated in table II below.

TABLE II

Substit- uent Spatial Configuration Entry R₅ R₆ C-6 C-8 C-9 C-10 C-13C-14 C-17  73 H H S S S R S S S  74 H H S R R R R R R  75 H H S S S R SS R  76 H H S R R R R R S  77 H — S S S R S S C═O  78 H — S R R R R RC═O  79 H H R R R R R R R  80 H H R S S R S S S  81 H H R S S R S S R 82 H H R R R R R R S  83 H — R S S R S S C═O  84 H — R R R R R R C═O 85 Me H S S S R S S S  86 Me H S R R R R R R  87 Me H S S S R S S R  88Me H S R R R R R S Cmpd 25 Me — S R S R S S C═O Cmpd 24 Me — R R S R S SC═O  91 Me H R R R R R R R  92 Me H S S S R S S S  93 Me H S R R R R R R 94 Me H S S S R S S R  95 Me H S R R R R R S  96 Me — R R S — R S C═O 97 Me — S R R R R R C═O Cmpd 5  Me H S R S R S S S  99 Me H R S S R S SS 100 Me H R S S S S S R 101 Me H R R R S R R S 102 Me — R S S R S S C═O103 Me — R R R R R R C═O 104 H H S S S R S S S 105 H H S R R R R R R 106H H S S S R S S R 107 H H S R R R R R S 108 H — S S S R S S C═O 109 H —S R R R R R C═O 110 H H R R R R R R R 111 H H R S S R S S S 112 H H R SS R S S R 113 H H R R R R R R S 114 H — R S S R S S C═O 115 H — R R R RR R C═O 116 Me H S S S R S S S 117 Me H S R R R R R R 118 Me H S S S R SS R 119 Me H S R R R R R S 120 Me — S S S S S S C═O 121 Me — S R R R R RC═O 122 Me H R R R R R R R 123 Me H R S S R S S S 124 Me H R S S S S S R125 Me H R R R S R R S 126 Me — R S S R S S C═O 127 Me — R R R R R R C═OCmpd 6  Me H R R S R S S S Cmpd 12 NH₂ H R R S S S S S Cmpd 13 NH₂ H S RS S S S S R₁, R₂, R₃, R₄: independently H, C₁-C₆ alkyl, substitutedalkyl, or halogen R₅: H, C₁-C₆ alkyl, substituted alkyl, sulfate, orglucuronide R₆: H, C₁-C₆ alkyl, or substituted alkyl, sulfate, orglucuronide, R₈: H, C₁-C₆ alkyl, or substituted alkyl, when  

  is a single bond; not present, when  

  is a double bond

The preferred compounds in Table II include compounds 5, 6, 12, 13, 24,and 25. At least one aspect of the instant invention is directed tothese preferred compounds, their method of use and making.

In yet another aspect of the invention, inventors illustrate thecompounds of the present invention in table III below:

TABLE III

Entry X Z T C-6 C-8 C-9 C-13 C-14 C-17 129 CH₂OH OH OH S S 130 CH₂OH OHOH S R 131 CH₂OH OH OH R R 132 CH₂OH OH OH R S 133 CH₂ONH₂ OH OH S S 134CH₂ONH₂ OH OH S R 135 CH₂ONH₂ OH OH R R 136 CH₂ONH₂ OH OH R S 137CH₂ONHMe OH OH S S 138 CH₂ONHMe OH OH S R 139 CH₂ONHMe OH OH R R 140CH₂ONHMe OH OH R S 141 CH₂ONMe₂ OH OH S S 142 CH₂ONMe₂ OH OH S R 143CH₂ONMe₂ OH OH R R 144 CH₂ONMe₂ OH OH R S 145 CH₂ONHAc OH OH S S 146CH₂ONHAc OH OH S R 147 CH₂ONHAc OH OH R R 148 CH₂ONHAc OH OH R S 149CH₂NH₂ OH OH S S 150 CH₂NH₂ OH OH S R 151 CH₂NH₂ OH OH R R 152 CH₂NH₂ OHOH R S 153 CH₂NHMe OH OH S S 154 CH₂NHMe OH OH S R 155 CH₂NHMe OH OH R R156 CH₂NHMe OH OH R S 157 CH₂NMe₂ OH OH S S 158 CH₂NMe₂ OH OH S R 159CH₂NMe₂ OH OH R R 160 CH₂NMe₂ OH OH R S 161 CH₂NHAc OH OH S S 162CH₂NHAc OH OH S R 163 CH₂NHAc OH OH R R 164 CH₂NHAc OH OH R S 165CH₂NHOH OH OH S S 166 CH₂NHOH OH OH S R 167 CH₂NHOH OH OH R R 168CH₂NHOH OH OH R S 169 CH₂NHOMe OH OH S S 170 CH₂NHOMe OH OH S R 171CH₂NHOMe OH OH R R 172 CH₂NHOMe OH OH R S 173 CH₂NHNH₂ OH OH S S 174CH₂NHNH₂ OH OH S R 175 CH₂NHNH₂ OH OH R R 176 CH₂NHNH₂ OH OH R S 177CH₂NHNHMe OH OH S S 178 CH₂NHNHMe OH OH S R 179 CH₂NHNHMe OH OH R R 180CH₂NHNHMe OH OH R S 181 CH₂NHNMe₂ OH OH S S 182 CH₂NHNMe₂ OH OH S R 183CH₂NHNMe₂ OH OH R R 184 CH₂NHNMe₂ OH OH R S 185 CH₂NHNHAc OH OH S S 186CH₂NHNHAc OH OH S R 187 CH₂NHNHAc OH OH R R 188 CH₂NHNHAc OH OH R S 189CH₂N(Me)—NH₂ OH OH S S 190 CH₂N(Me)—NH₂ OH OH S R 191 CH₂N(Me)—NH₂ OH OHR R 192 CH₂N(Me)—NH₂ OH OH R S 193 CH₂N(Me)—NHMe OH OH S S 194CH₂N(Me)—NHMe OH OH S R 195 CH₂N(Me)—NHMe OH OH R R 196 CH₂N(Me)—NHMe OHOH R S 197 CH₂N(Me)—NHAc OH OH S S 198 CH₂N(Me)—NHAc OH OH S R 199CH₂N(Me)—NHAc OH OH R R 200 CH₂N(Me)—NHAc OH OH R S 201 OCH₂NH₂ OH OH SS 202 OCH₂NH₂ OH OH S R 203 OCH₂NH₂ OH OH R R 204 OCH₂NH₂ OH OH R S 205OCH₂NHMe OH OH S S 206 OCH₂NHMe OH OH S R 207 OCH₂NHMe OH OH R R 208OCH₂NHMe OH OH R S 209 OCH₂NHAc OH OH S S 210 OCH₂NHAc OH OH S R 211OCH₂NHAc OH OH R R 212 OCH₂NHAc OH OH R S 213 NHCH₂OH OH OH S S 214NHCH₂OH OH OH S R 215 NHCH₂OH OH OH R R 216 NHCH₂OH OH OH R S 217NHCH₂OMe OH OH S S 218 NHCH₂OMe OH OH S R 219 NHCH₂OMe OH OH R R 220NHCH₂OMe OH OH R S 221 NHCH₂OAc OH OH S S 222 NHCH₂OAc OH OH S R 223NHCH₂OAc OH OH R R 224 NHCH₂OAc OH OH R S 225 NHCH₂NH₂ OH OH S S 226NHCH₂NH₂ OH OH S R 227 NHCH₂NH₂ OH OH R R 228 NHCH₂NH₂ OH OH R S 229NHCH₂NHMe OH OH S S 230 NHCH₂NHMe OH OH S R 231 NHCH₂NHMe OH OH R R 232NHCH₂NHMe OH OH R S 233 NHCH₂NMe₂ OH OH S S 234 NHCH₂NMe₂ OH OH S R 235NHCH₂NMe₂ OH OH R R 236 NHCH₂NMe₂ OH OH R S 237 NHCH₂NHAc OH OH S S 238NHCH₂NHAc OH OH S R 239 NHCH₂NHAc OH OH R R 240 NHCH₂NHAc OH OH R S 241N(Me)—CH₂OH OH OH S S 242 N(Me)CH₂OH OH OH S R 243 N(Me)CH₂OH OH OH R R244 N(Me)CH₂OH OH OH R S 245 N(Me)CH₂OMe OH OH S S 246 N(Me)CH₂OMe OH OHS R 247 N(Me)CH₂OMe OH OH R R 248 N(Me)CH₂OMe OH OH R S 249 N(Me)CH₂OAcOH OH S S 250 N(Me)CH₂OAc OH OH S R 251 N(Me)CH₂OAc OH OH R R 252N(Me)CH₂OAc OH OH R S 253 N(Me)CH₂NH₂ OH OH S S 254 N(Me)CH₂NH₂ OH OH SR 255 N(Me)CH₂NH₂ OH OH R R 256 N(Me)CH₂NH₂ OH OH R S 257 N(Me)CH₂NHMeOH OH S S 258 N(Me)CH₂NHMe OH OH S R 259 N(Me)CH₂NHMe OH OH R R 260N(Me)CH₂NHMe OH OH R S 261 N(Me)CH₂NMe₂ OH OH S S 262 N(Me)CH₂NMe₂ OH OHS R 263 N(Me)CH₂NMe₂ OH OH R R 264 N(Me)CH₂NMe₂ OH OH R S 265N(Me)CH₂NHAc OH OH S S 266 N(Me)CH₂NHAc OH OH S R 267 N(Me)CH₂NHAc OH OHR R 268 N(Me)CH₂NHAc OH OH R S Compound 18 OMe OH OH R R S S S SCompound 14 CH₂NHCH₂OCH₃ OH OH R R S S S S Compound 15 CH₂NHCH₂OCH₃ OHOH S R S S S S Compound 16 CH₂NHC(O)—OCH₃ OH OH R R S S S S Compound 17CH₂NHC(O)—OCH₃ OH OH S R S S S S Compound 19 (CH₂)₂OMe OH OH S R S S S SCompound 20 (CH₂)₄OMe OH OH R R S S S S Compound 21 (CH₂)₆OMe OH OH R RS S S S Compound 22 (CH₂)₈OMe OH OH R R S S S S Compound 30(CH₂)₄O(CH₂)₂—CH₃ OH OH R R S S S S Compound 31 (CH₂)₅O(CH₂)—CH₃ OH OH RR S S S S R₁, R₂, R₃, R₄: independently H, C₁-C₆ alkyl, substitutedalkyl, or halogen and  

  is a single or double bond; not present.

The preferred compounds in Table III include compounds 14, 15, 16, 17,18, 19, 20, 21, 22, and 31. At least one aspect of the instant inventionis directed to these preferred compounds, their method of use andmaking.

One specific non-limiting example for treatment of an identified cancerstate as described herein includes use a compound of formula IV such as:

The above active compounds may also be used as part of an in vitroassay, for example, in order to determine whether a candidate host islikely to benefit from treatment with the compound in question. Anyactive compound of the present invention may also be used as a standard,for example, in an assay, in order to identify other active compounds,other anti-proliferative agents, other anti-inflammatory agents, etc.

At least in one aspect of the instant invention, the candidate compoundswere evaluated for their estrogen receptor antagonistic activity. Theevaluation as to whether a compound is an estrogen receptor antagonistmay be carried out by various methodologies known in the art. In theinstant application, such capacity was determined by conducting theLuciferase binding assay according to the screening methods describedherein.

In a more preferred embodiment of this aspect of the invention, theestrogen receptor binding capacity were assessed by transientlytransfecting CV-1 cells with expression constructs for either ER(α) orER(β) plus an ERE-tk-luciferase reporter construct. The cells were thendivided into controls and candidate groups wherein the controls receivedno treatment, or were treated with estradiol alone (1 nM) and thecandidate groups received estradiol plus a compound of the invention atvarying concentrations. After 16-24 hours the cells were harvested andassayed for luciferase activity using a commercially available assaykit.

In yet another aspect of the instant invention, the IC₅₀ or the halfmaximal inhibitory concentration of the candidate compounds weredetermined to assess drug potency and potential dosing regimens for invivo use. One of ordinary skill in the art is readily able to ascertainsuch information using commonly known methodologies. As it has been welldescribed in the art, IC₅₀ represents and measures how much of aparticular substance/molecule is needed to inhibit some biologicalprocess by 50%. In the instant case, the IC₅₀ of the candidate compoundswere determined as the concentration that led to a response of 50%compared to the vehicle control cells.

As noted herein, the salts of the compounds of this invention refer tonon-toxic “pharmaceutically acceptable salts.” Other salts may, however,be useful in the preparation of the compounds according to the inventionor of their pharmaceutically acceptable salts. When the compounds of thepresent invention contain a basic group, salts encompassed within theterm “pharmaceutically acceptable salts” refer to non-toxic salts whichare generally prepared by reacting the free base with a suitable organicor inorganic acid. Representative salts include any such salt known inthe art. Where compounds of the present invention carry an acidicmoiety, suitable pharmaceutically acceptable salts thereof may includealkali metal salts, e.g., sodium or potassium salts; alkaline earthmetal salts, e.g., calcium or magnesium salts; and salts formed withsuitable organic ligands, e.g., quaternary ammonium salts.

To treat a mammalian subject, such as a human patient, an effectiveamount of one or more compounds of the present invention, or apharmaceutically-acceptable salt thereof, is administered to themammalian subject so as to promote exposure to or contact of cancercells or the targeted tumor growth. Effective dosage forms, modes ofadministration and dosage amounts may be determined empirically, andmaking such determinations is within the skill of the art. It isunderstood by the physician, veterinarian or clinician of ordinary skillin the art that the dosage amount will vary with the activity of theparticular compound employed, course and/or progression of the diseasestate, the route of administration, the rate of excretion of thecompound, renal and hepatic function of the patient, the duration of thetreatment, the identity of any other drugs being administered to thesubject, age, size and like factors well known in the medical arts. Asdiscussed herein, the compounds of the present invention can beadministered in such oral dosage forms as tablets, capsules (each ofwhich includes sustained release or timed release formulations), pills,powders, micronized compositions, granules, elixirs, tinctures,suspensions, syrups and emulsions. Likewise, they may also beadministered in intravenous (bolus or infusion), intraperitoneal,topical (e.g., ocular eyedrop), subcutaneous, intramuscular ortransdermal (e.g., patch) form, all using forms well known to those ofordinary skill in the pharmaceutical arts. Again, the ordinarily skilledphysician, veterinarian or clinician can readily determine and prescribethe effective amount of the drug required to prevent, counter or arrestthe progress of the condition.

Oral dosages of the present invention, when used for the indicatedeffects, will range between about 0.01 mg per kg of body weight per day(mg/kg/day) to about 100 mg/kg/day, preferably 0.01 to 10 mg/kg/day, andmost preferably 0.1 to 5.0 mg/kg/day. For oral administration, thecompositions are preferably provided in the form of tablets containing0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100 and 500milligrams of the active ingredient for the symptomatic adjustment ofthe dosage to the patient to be treated. A medicament typically containsfrom about 0.01 mg to about 500 mg of the active ingredient, preferably,from about 1 mg to about 100 mg of active ingredient. Intravenously, themost preferred doses will range from about 0.1 to about 10 mg/kg/minuteduring a constant rate infusion. Compounds of the present invention maybe administered in a single daily dose, or the total daily dosage may beadministered in divided doses of two, three or four times daily.

As noted herein, the compounds of the present invention can be used incombination with other anti-cancer agents or other agents which willenhance the treatment regime for the mammalian subject. The individualcomponents of such combinations can be administered separately atdifferent times during the course of therapy or concurrently in dividedor single combination forms to patients or regions of such patients inneed of such therapy. The instant invention is therefore to beunderstood as embracing all such regimes of simultaneous or alternatingtreatment and the term “administering” is to be interpreted accordingly.It will be understood that the scope of combinations of the compounds ofthis invention with other agents useful to treat the targeted cancercondition includes in principle any combination with any pharmaceuticalcomposition useful for treating disorders related to estrogenfunctioning.

It may be convenient or desirable to prepare, purify, and/or handle theactive compound in the form of a prodrug. The term “prodrug” as usedherein, pertains to a compound which, when metabolized, yields thedesired active compound or in itself is the active compound. Thisincludes for example adding a phosphoric acid ester moiety in suitablepositions such as positions 3, 6, 10 or 17. Typically, the prodrug isinactive, or less active than the active compound, but may provideadvantageous handling, administration, or metabolic properties. Forexample, some prodrugs are ethers of the active compound; duringmetabolism the ether group is cleaved to yield the active drug. Also,some prodrugs are activated enzymatically to yield the active compound,or a compound which, upon further chemical reaction, yields the activecompound. Thus, in the methods of treatment of the present inventiondisclosed herein, the term “administering” shall encompass the treatmentof the various conditions described with the compound specificallydisclosed or with a compound which may not be specifically disclosed,but which converts to the specified compound in vivo afteradministration to the patient. Metabolites of these compounds includeactive species produced upon introduction of compounds of this inventioninto the mammalian subject.

Compounds of the present invention may be prodrugs for potentanti-proliferative agents. Compounds which exhibit low or moderateintrinsic activity may act as prodrugs, and be metabolically activated(e.g., in vivo) to generate more potent compounds. This is especiallyuseful in cancer therapy where metabolic activation can be achieved byan enzyme that is expressed in tumors. Prodrugs, acting as a substrate,may be metabolized by CYP19, 17β-HSD, HS-demethylase or anothersteroidal linked enzyme to generate a potent anti-cancer agent. The (R)or (S)-6-methyloxoalkyl derivatives of exemestane suggest that it may beactive against numerous forms of cancer beyond breast cancer. Activityin inhibiting tumor cell growth in cells lines derived from breast,lung, colon, prostate, endometrial and ovarian cancers was observed forthe NDC-1011 enantiomer. For example, in vitro studies of tumor cellgrowth is the highest in cell lines that are CYP19 positive (MDA-MB-213and SK-OV-3) and is reduced in those cell lines that are CYP19 negative(MCF-7 and NIH:OVCAR-3) indicates that compound 24 may act as apro-drug.

While not bound by any theory, for example, if compound 24 is apro-drug, then any number of the body's normal steroidogenic enzymesshould be active towards compound 24 thereby converting compound 24 intothe active metabolite(s). This aspect of the invention can apply in thesame manner to both the S and the R diastereomers.

The prodrug compounds of the instant invention act in a manner analogousto that observed for endogenous androstonedione. Compound 24 isconverted to an aromatic ring by hydroxylation at the C-3 carbon ofcompound 24 via CYP19 to give rise to the metabolite compound 2.Compound 2 could undergo further hydroxylation at the C-17 carbon viathe reversible action of 17β-hydroxysteroid dehydrogenase (17β-HSD)resulting in the diol compound 4.

As with Estradiol, the diol compound 4 has an aromatic ring, but differsfrom estradiol with respect to the methyloxyalkyl substituent at the C-6carbon. The metabolism of compound 24 into the diol compound 4 couldoccur in any order. For example, compound 6 formed by 17β-HSD isconverted to compound 4 diol by CYP19 aromatization activity.

Without being bound to any theories, it has been reported that Estradiolbinds to the receptor ligand pocket of estrogen receptors (both ERα andERβ), via the C17-OH (via His 524); and the C3-OH (via Arg 394 and Glu353). As with Estradiol, binding of compound 4 diol in the same ligandpocket of ERα and ERβ via similar amino acid bindings may occur.Additionally, the presence of the methyloxyalkyl substituent at the C-6carbon of compound 4 may alter the conformation of the normalligand-bound receptor resulting in modified activity accounting for theobserved anti-tumor activity.

In addition, demethylase enzyme activity directed at the C-6 methylgroup of compound 24 (or one of the metabolites of compound 24), mayindicate the formation of triol metabolite compound 8. With alcoholgroups at the C-3, C-6 and C-17 carbons, such an compound 8 triolmetabolite may bind to a broad spectrum of steroid receptors in a rangeof tissues involving various combinations of the C-3, C-6 and C-17alcohols.

Compositions

As used herein, the term “composition” is intended to encompass aproduct comprising the specified ingredients in the specified amounts,as well as any product which results, directly or indirectly, fromcombination of the specified ingredients in the specified amounts.

Pharmaceutical formulations of the present invention include thosesuitable for oral, nasal, topical (including buccal and sublingual),rectal, vaginal and/or parenteral administration. Regardless of theroute of administration selected, the active ingredient(s) areformulated into pharmaceutically-acceptable dosage forms by conventionalmethods known to those of skill in the art.

The amount of the active ingredient(s) which will be combined with acarrier material to produce a single dosage form will vary dependingupon the host being treated, the particular mode of administration andall of the other factors described above. The amount of the activeingredient(s) which will be combined with a carrier material to producea single dosage form will generally be that amount of the activeingredient(s) which is the lowest dose effective to produce atherapeutic effect.

Methods of preparing pharmaceutical formulations or compositions includethe step of bringing the active ingredient(s) into association with thecarrier and, optionally, one or more accessory ingredients. In general,the formulations are prepared by uniformly and intimately bringing theactive ingredient(s) into association with liquid carriers, or finelydivided solid carriers, or both, and then, if necessary, shaping theproduct.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or nonaqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of the activeingredient(s). The active ingredient(s) may also be administered as abolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theprodrug(s), active ingredient(s) (in their micronized form) is/are mixedwith one or more pharmaceutically-acceptable carriers, such as sodiumcitrate or dicalcium phosphate, and/or any of the following: (1) fillersor extenders, such as starches, lactose, sucrose, glucose, mannitol,and/or silicic acid; (2) binders, such as, for example,carboxymethyl-cellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; (3) humectants, such as glycerol; (4)disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and sodium carbonate;(5) solution retarding agents, such as paraffin; (6) absorptionaccelerators, such as quaternary ammonium compounds; (7) wetting agents,such as, for example, cetyl alcohol and glycerol monostearate; (8)absorbents, such as kaolin and bentonite clay; (9) lubricants, such astalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents.In the case of capsules, tablets and pills, the pharmaceuticalcompositions may also comprise buffering agents. Solid compositions of asimilar type may also be employed as fillers in soft and hard-filledgelatin capsules using such excipients as lactose or milk sugars, aswell as high molecular weight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered activeingredient(s) moistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient(s) thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter. These compositions mayalso optionally contain opacifying agents and may be of a compositionthat they release the active ingredient(s) only, or preferentially, in acertain portion of the gastrointestinal tract, optionally, in a delayedmanner. Examples of embedding compositions which can be used includepolymeric substances and waxes. The active ingredient(s) can also be inmicroencapsulated form.

Liquid dosage forms for oral administration of the active ingredient(s)include pharmaceutically-acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient(s), the liquid dosage forms may contain inert diluentscommonly used in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethylacetate, butyl alcohol, benzyl benzoate, propylene glycol,glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive,castor and sesame oils), glycerol, amyl alcohol, tetrahydrofurylpolyethylene glycols and fatty acid esters of sorbitan, and mixturesthereof.

Besides inert diluents the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents. Suspensions, inaddition to the active ingredient(s), may contain suspending agents as,for example, ethoxylated alcohols, polyoxyethylene sorbitol and sorbitanesters, microcrystalline cellulose, aluminum metahydroxide, bentonite,agar-agar and tragacanth, and mixtures thereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing the active ingredient(s) with one ormore suitable nonirritating excipients or carriers comprising, forexample, cocoa butter, polyethylene glycol, wax or salicylate and whichis solid at room temperature, but liquid at body temperature and,therefore, will melt in the rectum or vaginal cavity and release theactive ingredient(s). Formulations of the present invention which aresuitable for vaginal administration also include pessaries, tampons,creams, gels, pastes, foams or spray formulations containing suchcarriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration of the activeingredient(s) include powders sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The activeingredient(s) may be mixed under sterile conditions withpharmaceutically-acceptable carrier, and with any buffers, orpropellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to theactive ingredient(s), excipients, such as animal and vegetable fats,oils, waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof. Powders and sprays can contain, in additionto the active ingredient(s), excipients such as lactose, talc, silicicacid, aluminum hydroxide, calcium silicates and polyamide powder, ormixtures of these substances. Sprays can additionally contain customarypropellants such as chlorofluorohydrocarbons and volatile unsubstitutedhydrocarbons, such as butane and propane.

Compounds of the present invention may be administered in intranasalform via topical use of suitable intranasal vehicles, or via transdermalroutes, using those forms of transdermal skin patches well known tothose of ordinary skill in the art. A transdermal delivery systemprovides for continuous administration throughout the dosage regimen.Transdermal patches have the added advantage of providing controlleddelivery of the active ingredient(s) to the body. Such dosage forms canbe made by dissolving, dispersing or otherwise incorporating the activeingredient(s) in a proper medium, such as an elastomeric matrixmaterial. Absorption enhancers can also be used to increase the flux ofthe active ingredient(s) across the skin. The rate of such flux can becontrolled by either providing a rate-controlling membrane or dispersingthe active ingredient(s) in a polymer matrix or gel.

The compounds of the present invention can also be administered in theform of liposome delivery systems, such as small unilamellar vesicles,large unilamellar vesicles and multilamellar vesicles. Liposomes can beformed from a variety of phospholipids, such as cholesterol,stearylamine or phosphatidylcholines.

Another mode of delivery for the compounds of the present invention maybe delivery via the use of monoclonal antibodies as individual carriersto which the compound molecules are coupled. The compounds of thepresent invention may also be coupled with soluble polymers astargetable drug carriers. Such polymers can includepolyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamide-phenol, polyhydroxy-ethylaspartamide-phenol, orpolyethyleneoxide-polylysine substituted with palmitoyl residues.Furthermore, the compounds of the present invention may be coupled to aclass of biodegradable polymers useful in achieving controlled releaseof a drug, for example, polylactic acid, polyglycolic acid, copolymersof polyactic and polyglycolic acid, polyepsilon caprolactone,polyhydroxy butyric acid, polyorthoesters, polyacetals,polydihydropyrans, polycyanoacrylates and crosslinked or amphipathicblock copolymers of hydrogels.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise the active ingredient(s) in combination with oneor more pharmaceutically-acceptable sterile isotonic aqueous ornonaqueous solutions, suspensions or emulsions, or sterile powders whichmay be reconstituted into sterile injectable solutions or dispersionsjust prior to use, which may contain antioxidants, buffers, soluteswhich render the formulation isotonic with the blood of the intendedrecipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size, and by the use of surfactants.

These compositions may also contain adjuvants such as wetting agents,emulsifying agents and dispersing agents. It may also be desirable toinclude isotonic agents, such as sugars, sodium chloride, and the likein the compositions. In addition, prolonged absorption of the injectablepharmaceutical form may be brought about by the inclusion of agentswhich delay absorption such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of the activeingredient(s), it is desirable to slow the absorption of the drug fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material havingpoor water solubility. The rate of absorption of the activeingredient(s) then depends upon its/their rate of dissolution which, inturn, may depend upon crystal size and crystalline form. Alternatively,delayed absorption of parenterally-administered active ingredient(s) isaccomplished by dissolving or suspending the active ingredient(s) in anoil vehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe active ingredient(s) in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of the activeingredient(s) to polymer, and the nature of the particular polymeremployed, the rate of release of the active ingredient(s) can becontrolled. Examples of other biodegradable polymers includepoly(orthoesters) and poly(anhydrides). Depot injectable formulationsare also prepared by entrapping the active ingredient(s) in liposomes ormicroemulsions which are compatible with body tissue. The injectablematerials can be sterilized for example, by filtration through abacterial-retaining filter.

Preferably the composition delivered in the form of an injectable dosageform comprise a biocompatible polymer, a compatible form of thepresently disclosed compounds and a biocompatible solvent whichsolubilizes the biocompatible polymer wherein the weight percents of thebiocompatible polymer, the instant and biocompatible solvent are basedon the total weight of the complete composition; further whereinsufficient amounts of said polymer are employed in said composition suchthat, upon delivery to a vascular site, the polymer is able toprecipitate and allow release of the active compound in doses sufficientto stop tumor growth.

Still another aspect of this embodiment would observe for appropriateviscosity of said composition, preferably in the range of about 10 to200 cSt at 40° C.

More preferably, the composition delivered locally to the solid tumorcomprises a biocompatible polymer at a concentration of from about 1 to95 weight percent, active compound at a concentration of from about 5 toabout 75 weight percent, and a biocompatible solvent from about 5 toabout 95 weight percent, wherein the weight percent of the allcomponents is based on the total weight of the complete composition andfurther wherein the composition has a viscosity of at least 10 to about200 and more preferably at least about 200 cSt at 40° C.

Biodegradable polymers are disclosed in the art. For example, Dunn, etal. in U.S. Pat. No. 4,938,763, discloses the following examples ofbiodegradable polymers: linear-chain polymers such as polylactides,polyglycolides, polycaprolactones, polyanhydrides, polyamides,polyurethanes, polyesteramides, polyorthoesters, polydioxanones,polyacetals, polyketals, polycarbonates, polyorthocarbonates,polyphosphazenes, polyhydroxybutyrates, polyhydroxyvalerates,polyalkylene oxalates, polyalkylene succinates, poly(malic acid),poly(amino acids), polyvinylpyrrolidone, polyethylene glycol,polyhydroxycellulose, chitin, chitosan, and copolymers, terpolymers andcombinations thereof. Other biodegradable polymers include, for example,gelatin, collagen, etc.

Suitable non-biodegradable biocompatible polymers include, by way ofexample, cellulose acetates, ethylene vinyl alcohol copolymers,hydrogels (e.g., acrylics), polyacrylonitrile, polyvinylacetate,cellulose acetate butyrate, nitrocellulose, copolymers ofurethane/carbonate, copolymers of styrene/maleic acid, and mixturesthereof.

Preferred biocompatible polymers can include acrylic polymers, cellulosediacetate and ethylene vinyl alcohol copolymer, polyethylene glycol,chitosen, collagen and gelatin. Such polymers are either commerciallyavailable or can be prepared by art recognized procedures. In apreferred embodiment, the number average molecular weight, as determinedby gel permeation chromatography composition is from about 5,000 toabout 200,000 more preferably from about 25,000 to about 180,000 andstill more preferably from about 50,000 to 100,000.

It is still another aspect of this invention to employ a biocompatiblecontrast agent within the composition to observe and monitor theclinical progress of the local site of interest. These contrast agentsinclude water soluble contrast agents and water insoluble contrastagents. Preferably, the water insoluble contrast agent is abiocompatible material selected from the group consisting of bariumsulfate, tantalum powder and tantalum oxide. In still a furtherpreferred embodiment, the biocompatible solvent is water,dimethylsulfoxide (DMSO), ethanol, ethyl lactate or acetone.

The formulations may be presented in unit-dose or multi-dose sealedcontainers, for example, ampoules and vials, and may be stored in alyophilized condition requiring only the addition of the sterile liquidcarrier, for example water for injection, immediately prior to use.Extemporaneous injection solutions and suspensions maybe prepared fromsterile powders, granules, nanoparticles and tablets of the typedescribed above.

The pharmaceutical compositions of the present invention may also beused in the form of veterinary formulations, including those adapted forthe following: (1) oral administration, for example, drenches (aqueousor nonaqueous solutions or suspensions), tablets, boluses, powders,granules or pellets for admixture with feed stuffs, pastes forapplication to the tongue; (2) parenteral administration, for “ampule,by subcutaneous, intramuscular or intravenous injection as, for example,a sterile solution or suspension or, when appropriate, by intramammaryinjection where a suspension or solution is introduced into the udder ofthe animal via its teat; (3) topical application, for example, as acream, ointment or spray applied to the skin; or (4) intravaginally, forexample, as a pessary, cream or foam or any other methods fit to bythose of ordinary skill in the art for administration to a region ofinterest.

Although the present invention has been described with reference tocertain embodiments, one skilled in the art will appreciate that thepresent invention can be practiced by other than the describedembodiments, which have been presented for purposes of illustration andnot of limitation. Therefore, the scope of the appended claims shouldnot be limited to the description of the embodiments contained herein.

The general methods given in Schemes 1, 2, and 3 for the preparation ofcompounds exemplified in formulas I—IV and in Tables I, II, and IIIabove are further illustrated by the following examples. Specifically,the methods given in Schemes 1 and 2 for the preparation of6-alkoxyalkyl Estradiol compounds are illustrated by Examples 1-5 shownbelow, and Scheme 3 is for the preparation of 6-amino derivatives ofestradiol. An example of assessing the estrogen receptor bindingcapacity is articulated in example 4, and finally assessing the IC₅₀ ofthe preferred compounds of the instant invention and their comparativeefficacy is given in example 5. Unless otherwise specified all startingmaterials and reagents are of standard commercial grade, and are usedwithout further purification, or are readily prepared from suchmaterials by routine methods. Those skilled in the art of organicsynthesis will recognize that starting materials and reaction conditionsmay be varied to achieve the desired end product.

Example 1

Methods of Preparing 6-hydroxymethyl-androsta-1,4-diene-3,17 dione.

In a reaction system, sufficient amounts of(+)androsta-1,4-diene-3,17-dione (ADD), 12.2 equivalents pyrrolidine,catalytic acetic acid, denatured ethanol (95/5 ethanol/methanol) and6-7% tetrahydrofuran (THF) are heated to 30 to 40° C. for a minimum of16 hours to form 1,3-dipyrrolidinoandrosta-3,5-diene-17one. Once the ADDcontent reaches to a less than 3% by HPLC area, or it becomes static orthe resulting dipyrrolidinoandrostadiene begins to revert to ADD, thereaction mixture is cooled to 5±5° C. The resulting compound is thencollected and washed with cold denatured ethanol. Yields are typically70-80% on a dry basis with purities typically 90-95% by HPLC areapercent.

The resulting 1,3-dipyrrolidinoandrosta-3,5-diene-17one is then mixed inamount of 1 equivalent with 2.6 equivalents formalin (formaldehyde) in10 ml dichloromethane/g at room temperature. The reaction mixture isthen acidified to a pH of about 2 with 2% sulfuric acid solution.Accordingly, an organic layer is formed, which is washed with 2%sulfuric acid and 1:1 water/brine. Solvent exchange into toluene(approximately 10 ml/g) is then carried out wherein the productcrystallizes as toluene exchange transpires. Said product is collectedwashed and dried to provide 6-hydroxymethyl-androsta-1,4-diene-3,17dione. One of ordinary skill in the art can further modify thestereochemistry at position 6, if so desired, by employing knowntechniques in the art.

Example 2

Methods of Preparing Compounds 3 and 4.

As outlined in Scheme 2, estradiol compounds 3 and 4 are synthesized inthe following manner. The protected estradiol is prepared by reaction ofβ-estradiol with dihydropyran in THF, using toluenesulfonic acid orcamphorsulfonic acid as catalyst. As one of ordinary skill in the artcan appreciate, this reaction is an equilibrium reaction and would notgo to completion under such conditions. Thus, both the mono-protectedestradiols can be found in the reaction mixture. Such crude reactionmixture would undergo a trituration step with acetonitrile causing thedesired bis-THP estradiol to crystallize in approximately 70% yield.

As shown in Scheme 2, the intermediate aldehyde is obtained viaacylation at the benzylic 6-position with a strong base mixture referredto as LiDAKOR: butyl lithium, diisopropylamine, and potassiumtert-amylate. Under such conditions at −70° C., one of ordinary skill inthe art can appreciate the abstraction of a proton at a benzylicposition. The intermediate aldehyde is then purified by columnchromatography to give a syrup in approximately 50% yield. Reduction ofthe aldehyde with an excess of lithium aluminum hydride results in highyields of the racemic hydroxymethyl estradiol compound as a glassy foam.

For purposes of preparing compounds 3 and 4, the methoxymethylintermediate compound is prepared by methylation of the racemichydroxymethyl estradiol compound with sodium hydride and methyl iodide.The methoxymethyl intermediate is purified by column chromatography togive a glassy foam. Deprotection of the protecting groups givesdeprotected racemic 6-methoxymethyl estradiol. Separation of theenantiomers is performed using chiral preparative HPLC to give thecompounds 3 and 4. For compound 4, a chiral purity of >95:5 R:S isrealized. For compound 3, a chiral purity of 86:14 S:R is realized. Itis well within the level of one of ordinary skill in the art to employNMR for determination of the absolute stereochemistry of the 6-position,where the 4- and 6-protons are diagnostic.

Example 3

Methods of Preparing Compounds 7 and 8.

Using the same methodologies described in Example 2, the racemichydroxymethyl estradiol compound is synthesized. Deprotection of thesame is then achieved with catalytic hydrogen chloride in methanol, andthe resulting racemic triol is separated on chiral preparative HPLC togive two fractions, one enriched for compound 7 and the other enrichedfor compound 8. For each compound, chiral purity of >95:5 R:S and S:R isrealized respectively. Absolute stereochemistry of the 6-position isestablished by NMR, where the 4- and 6-protons are diagnostic.

Example 4

Methods of Preparing Compound 10.

Using the same methodologies as in examples 1-2, compound 4 is prepared.To a solution of compound 4 (0.32 g, 1 mmol) in DCM (30 ml), aceticanhydride (0.6 ml, 6 mmol, 3 eq), TEA (0.5 ml, 3.6 mmol, 1.8 eq) andDMAP (50 mg) is added. The reaction solution is stirred at ambienttemperature for 3 hrs. Thin Layer Chromatography (“TLC”) follows thereaction to completion.

The reaction solution is washed with 1M HCl (20 ml), saturated NaHCO₃(20 ml) and brine (20 ml), respectively. The DCM phase is dried overMgSO₄ and filtered. The filtrate is evaporated and dried in high vacuumat 60° C. for 3 hrs and at ambient temperature overnight to afford puredesired protected methoxy compound of Scheme 3 (0.4 g, white,quantitative).

To a solution of protected methoxy compound of Scheme 3 (0.35 g, 0.87mmol) in DCM is added iodotrimethylsilane (6 ml, 44 mmol, 50 eq) atambient temperature. The yellow solution is stirred at 38° C. overnightunder argon.

The reaction solution is cooled in an ice-bath. Excess sat. NaHCO₃ (10ml) is slowly added to quench the reaction. After separation of themixture, the organic phase is washed with brine and concentrated forsilica gel purification using 3% MeOH in DCM as mobile phase. A yield(80%) of 270 mg of pure hydroxyl compound of Scheme 3 is obtained.

A solution of hydroxyl compound of Scheme 3(250 mg, 0.65 mmol),triphenylphosphine (222 mg, 0.85 mmol, 1.3 eq), and N-hydroxyphthalimide(140 mg, 0.85 mmol, 1.3 eq) in THF (20 ml) is cooled in an ice-batch. Tothe cooled solution is added diethyl azodicarboxylate (0.6 ml, 1 mmol,1.5 eq). The reaction mixture is warmed to ambient temperature andstirred overnight.

The reaction mixture is then evaporated, and the resulting residue isdiluted with DCM (50 ml), washed with brine and concentrated for silicagel purification using 3% MeOH/DCM as mobile phase to yield 260 mg (75%)of desired white phthalimide compound of Scheme 3.

A solution of phthalimide compound of Scheme 3(580 mg, 1.1 mmol) inanhydrous DCM (30 ml) is cooled in an ice-batch. Methyl hydrazine (0.22ml, 2.2 mmol, 4 eq) is added. The mixture is warmed to ambienttemperature and stirred for 3 hrs. LC-MS follows the reaction tocompletion.

The reaction solution is washed with a solution of brine and sat. NaHCO₃(1:1, 10 ml). The aqueous phase is washed with DCM (10 ml). The combinedDCM phases are evaporated to dryness under high vacuum. LC-MS confirmsthat the crude product contains two major aminooxy products of Scheme 3.

The crude mixture (˜0.7 mmol) of aminooxy compounds of Scheme 3 in MeOH(60 ml) is cooled in an ice-bath. A solution of sodium carbonate (0.5 g,4.7 mmol) in water (10 ml) and a solution of NaOH (0.8 g, 20 mmol) inwater (10 ml) is subsequently added. The reaction is warmed to ambienttemperature and stirred overnight.

The pH of final reaction solution is about 14. A solution of sat. sodiumbicarbonate (˜10 ml) is added to adjust pH to 10. The mixture is thenevaporated to remove most of the methanol. To the resulting mixture isadded DCM (100 ml) and sat. sodium bicarbonate (30 ml) for extraction.The aqueous phase is washed with DCM (2×50 ml). The combined DCM phasesare evaporated to dryness to give crude mixture 400 mg.

The crude mixture is purified by silica gel column using 5% MeOH/DCM asmobile phase to afford desired final product compound 10 (135 mg, offwhite, 60% yield, 98% HPLC purity, NMR and LC-MS confirmed).

Example 5

Methods of Preparing Compound 21

a)(8R,9S,13S,14S,17S)-3,17-bis(methoxymethoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene—Chloromethylmethyl ether (7.0 mL, 92.0 mmol) is added to a solution of β-estradiol(5 g, 18.4 mmol) and diisopropylethylamine (16.0 mL 92 mmol) in 100 mLof THF. The reaction mixture is heated to reflux and stirred for 18hours. The THF is removed in vacuo, and the yellow/brown oil ispartitioned between water and CH₂Cl₂. The organic layer is separated,washed with brine, dried (Na₂SO₄), filtered, and evaporated in vacuo togive a golden oil. Purification by silica gel column chromatography (10%EtOAc/Hex) affords the title compound as a viscous, clear oil (5.7 g,86%).

b)(8R,9S,13S,14S,17S)-3,17-bis(methoxymethoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-6-ol—Toa solution of potassium tert-butoxide (8.87 g, 79.0 mmol) anddiisopropylamine (11.2 mL, 79.0 mmol) in 80 mL of anhydrous THF cooledto −78° C. under argon is added n-butyllithium (49.4 mL, 79.0 mmol, 1.6M in hexane) dropwise. The reaction mixture is stirred at −78° C. for30-45 minutes. A solution of the compound from a) (5.7 g, 15.8 mmol) in45 mL of THF is then added dropwise, and the reaction mixture is stirredfor 3 hours at −78° C. During the addition of the compound from a), thereaction turns a deep red color. Trimethyl borate (10.6 mL, 94.8 mmol)is then added slowly, and the mixture is warmed to 0° C. and stirred for2 hours. Hydrogen peroxide (24 mL of a 30% aq. solution) is then added,and the reaction mixture is warmed to room temperature and stirred for afurther 1 hour. The reaction is cooled back to 0° C. and carefullyquenched with a 10% aq. Na₂S₂O₃ solution (70 ml). The resulting mixtureis extracted with EtOAc (2×), and the combined organic extracts aredried (Na₂SO₄), filtered, and evaporated in vacuo to give a yellow/brownoil. Purification by silica gel column chromatography (25% EtOAc/Hex)affords the title compound as a white solid (3.5 g, 59%).

c)(8R,9S,13S,14S,17S)-3,17-bis(methoxymethoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-6-one—Dess-MartinPeriodinane (9.46 g, 22.3 mmol) is added portionwise to a solution ofthe compound from b) (7.0 g, 18.6 mmol) in 300 mL of CH₂Cl₂. Theresulting reaction mixture stirred at room temperature for 3 hours. Themixture is poured into water and the layers are separated. The aqueouslayer is extracted with CH₂Cl₂, and the combined organic extracts arewashed with brine, dried (Na₂SO₄), filtered, and evaporated in vacuo togive a gooey, brown solid. Purification by silica gel columnchromatography (15% EtOAc/Hex) affords the title compound as a paleyellow, viscous oil (6.0 g, 86%).

d) ethyl2-(((8R,9S,13S,14S,17S)-3,17-bis(methoxymethoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-6-ylidene)acetate—Triethylphosphonoacetate (4.1 mL, 20.8 mmol) is added to a mixture of sodiumhydride (832 mg, 20.8 mmol) in 25 mL of THF at room temperature. Afterapproximately 10 minutes, a solution of the compound from c) (3.9 g,10.4 mmol) in 10 mL of THF is added dropwise. The resulting reactionmixture is heated to reflux in a sealed tube for 72 hours. The mixtureis concentrated in vacuo and purified by silica gel columnchromatography (gradient from 5% EtOAc/Hex to 40% EtOAc/Hex) to give thetitle compound as a clear, viscous oil (3.4 g, 74%).

e)2-((8R,9S,13S,14S,17S)-3,17-bis(methoxymethoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-6-ylidene)ethanol—Asolution of the compound from d) (3.1 g, 6.97 mmol) in 65 mL of THF istreated with lithium aluminum hydride (5.2 mL, 10.46 mmol, 2 M in THF)dropwise at 0° C. The cold bath is removed, and the reaction mixture isstirred at room temperature for 15 minutes. The reaction is cooled backto 0° C. and quenched by the careful addition of 1.3 mL of water,followed by 2.6 mL of 2N NaOH, and then 1.3 mL of water. The mixture isstirred vigorously until a white solid forms. The mixture is filtered,and the filtrate is concentrated in vacuo to give the title compound asa clear oil (2.8 g, 99%).

f)2-((6S,8R,9S,13S,14S,17S)-3,17-bis(methoxymethoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-6-yl)acetaldehyde—Amixture of the compound from e) (3.09 g, 7.68 mmol) and 10% Pd/C (500mg) in 100 mL of ethyl acetate is stirred under 40 psi of H₂ (g) for 5hours at room temperature. The mixture is filtered through Celite, andthe Celite is washed well with ethyl acetate. The filtrate isconcentrated in vacuo to give a pale yellow oil (3.1 g). The oil isdissolved in 100 mL of dichloromethane, and Dess-Martin Periodinane (3.9g, 9.22 mmol) is added portionwise. The resulting reaction mixture isstirred at room temperature for 30 minutes. The mixture is poured intowater and extracted with CH₂Cl₂. The combined organic extracts arewashed with brine, dried (Na₂SO₄), filtered, and evaporated in vacuo togive a brown solid. Purification by silica gel column chromatography(15% EtOAc/Hex) affords the title compound as a clear oil (2.0 g, 65%).

g)4-((6R,8R,9S,13S,14S,17S)-3,17-bis(methoxymethoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-6-yl)but-2-en-1-ol—Lithiumbis(trimethylsilyl)amide (18.4 mL, 18.4 mmol, 1.0 M in THF) is addeddropwise to a suspension of (2-hydroxyethyl) triphenylphosphoniumbromide (3.37 g, 8.70 mmol) in 60 mL of THF at 0° C. After 1 hour, thegolden brown solution is treated with a solution of the compound from f)(1.4 g, 3.48 mmol) in 10 mL of THF dropwise. The resulting reactionmixture is stirred at 0° C. for 40 minutes and then quenched withsaturated aqueous NH₄Cl. The resulting mixture is extracted with EtOAc(2×), and the combined organic extracts are dried (Na₂SO₄), filtered,and evaporated to give a brown oil. Purification by silica gel columnchromatography (gradient from 20% EtOAc/Hex to 75% EtOAc/Hex) affordsthe title compound as a yellow, viscous oil (680 mg, 45%).

h)4-((6R,8R,9S,13S,14S,17S)-3,17-bis(methoxymethoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-6-yl)but-2-enal—Dess-MartinPeriodinane (437 mg, 1.03 mmol) is added to a solution of the compoundfrom g) (370 mg, 0.86 mmol) in 15 mL of CH₂Cl₂ at room temperature. Theresulting reaction mixture is stirred for 10 minutes and then pouredinto water. The layers are separated and the aqueous layer is extractedwith CH₂Cl₂ (2×). The combined organic extracts are washed with brine,dried (Na₂SO₄), filtered, and evaporated in vacuo to give a brown oil.Purification by silica gel column chromatography (gradient from 5%EtOAc/CH2Cl2 to 10% EtOAc/CH₂Cl₂) affords the title compound as a paleyellow, viscous oil (358 mg, 86%).

i)6-((6R,8R,9S,13S,14S,17S)-3,17-bis(methoxymethoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-6-yl)hexa-2,4-dien-1-ol—Lithiumbis(trimethylsilyl)amide (4.3 mL, 4.29 mmol, 1.0 M in THF) is addeddropwise to a suspension of (2-hydroxyethyl) triphenylphosphoniumbromide (786 mg, 2.03 mmol) in 14 mL of THF at 0° C. After 30 minutes,the golden brown solution is treated with a solution of the compoundfrom h) (345 mg, 0.81 mmol) in 2 mL of THF dropwise. The resultingreaction mixture is stirred at 0° C. for 20 minutes and quenched withsaturated aqueous NH₄Cl. The resulting mixture is extracted with EtOAc(2×), and the combined organic extracts are dried (Na₂SO₄), filtered,and evaporated to give a brown oil. Purification by silica gel columnchromatography (gradient from 5% EtOAc/CH₂Cl₂ to 40% EtOAc/CH₂Cl₂)affords the title compound as a yellow, viscous oil (140 mg, 38%).

j)(6R,8R,9S,13S,14S,17S)-6-(6-methoxyhexa-2,4-dien-1-yl)-3,17-bis(methoxymethoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene—Asolution of the compound in i) (135 mg, 0.3 mmol) is cooled to 0° C.,and sodium hydride (120 mg, 3.0 mmol) is added portionwise. After 5-10minutes, iodomethane (0.19 mL, 3.0 mmol) is added dropwise, and theresulting reaction mixture is warmed to room temperature and stirred for4 hours. EtOAc is added and the reaction is carefully quenched withwater. The layers are separated and the organic layer is dried (Na₂SO₄),filtered, and evaporated to give a brown oily residue. Purification bysilica gel column chromatography (gradient from 5% EtOAc/Hex to 20%EtOAc/Hex) affords the title compound as a clear oil (92 mg, 65%).

k)(6R,8R,9S,13S,14S,17S)-6-(6-methoxyhexyl)-3,17-bis(methoxymethoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene—Amixture of the compound in j) (90 mg, 0.19 mmol) and 10% Pd/C (100 mg)in 5-10 mL of ethyl acetate is stirred under a balloon of H₂ (g) for 16hours at room temperature. The mixture is filtered through Celite, andthe Celite is washed well with ethyl acetate. The filtrate isconcentrated in vacuo to give the title compound as a clear oil (90 mg,99%).

l)(6R,8R,9S,13S,14S)-6-(6-methoxyhexyl)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diol(Compound 21)—A solution of the compound from k) (90 mg, 0.19 mmol) in1.5 mL each of 6 N HCl and THF is stirred for 5 hours at roomtemperature. The reaction mixture is diluted with water and extractedwith EtOAc (2×). The combined organic extracts are dried (Na₂SO₄),filtered, and evaporated in vacuo to give a clear, oily residue.Purification by silica gel column chromatography (gradient from CH2Cl2to 30% EtOAc/CH2Cl2) afforded Compound 21 as a white solid foam (38 mg,52%).

Example 6

Methods of Preparing Compounds 30 and 31

Compounds 30 and 31 are prepared according to Scheme 4 (actually yieldsare provided).

Example 7

Methods of Determining Estrogen Receptor Binding Capacity UsingLuciferase Activity.

Estrogen receptor-negative CV-1 kidney cells are maintained inDulbecco's modified Eagle's medium with 4.5 g/L glucose supplementedwith 10% fetal bovine serum and 100 units/ml penicillin-streptomycin at37° C. in a humidified 5% CO₂ atmosphere. The cells are then plated in6-well dishes at a density of 2×10⁵ cells per well in phenol-red freeDulbecco's modified Eagle's medium containing 10%charcoal-dextran-stripped fetal bovine serum. CV-1 cells are transfectedusing LipofectAMINE reagent according to the manufacturer's protocol.Transfections containing 1.5 ug of reporter plasmid (containingERE-tk-luciferase containing a single ERE cloned upstream of thethymidine kinase promoter and luciferase gene) and 0.5 ug of either ERαor ERβ expression vector (containing CMV-ERα or CMV-ER_(β) full lengthcoding sequence respectively). The next day, cells receive no treatment(controls) or are treated with estradiol alone (1 nM) or estradiol plusa compound of the invention (at varying concentrations). After 16-24hours, cells are harvested and assayed for luciferase activity.

At the outset, cell monolayers are washed twice with ice-coldphosphate-buffered saline and incubated for 15 minutes in 250 μl of 1×cell culture lysis reagent (Promega, Madison, Wis.). Cell extracts aretransferred to a fresh tube and assayed using the luciferase assaysystem (Promega). For each assay, 10 μl of extract is diluted with 90 μlof 1× cell culture lysis reagent. Luminescence is read using anAutoLumat LB953 luminometer.

A compound or a salt thereof, which is identified by the binding assaydescribed herein, is a compound that inhibits the binding of estrodialat the ligand binding site of the estrogen receptors. Specifically, itis a compound or a salt thereof that is envisioned to cause cellproliferation statasis and accordingly exerts its pharmacologicalactivity.

CV-1 cells are transfected with two plasmid constructs, the reporterconstruct ERE-tk-luciferase and a CMV-ER-β construct. Transfectedcontrol (Ctrl) CV-1 cells receive no treatment while estradiol treatedcells receive estradiol E2) added alone at 10⁻⁹ M (1 nM). In the case ofthe compounds of the invention, each compound respectively is eitheradded alone at 10⁻⁸ M (10 nM) or at 10⁻⁸ M plus 10⁻⁹ M estradiol (E2).

Example 8

Method of Determining the IC₅₀ Values of the Candidate Compounds.

The cell lines listed are maintained at approximately 5% CO₂, 37° C.,95% relative humidity in the media appropriate for that cell line. Thecells are sub-cultured every two to three days and plated in clearbottom 96-well plates at a density of 1×10⁴ cells/well and incubated atca. 5% CO₂, 37° C. overnight prior to initiation of the assay. To begincell viability assays, the media in the cell plate (100 μL) is replacedwith fresh media (100 μL). The test articles are serially diluted 1:2 infresh media in duplicate and added to the cells (100 μL) at final sampleconcentrations of 0.46, 1.37, 4.12, 12.35, 37.04, 111.1, 333.3 and 1000μM (≤1% DMSO) in a total volume of 200 μL. Wells containing no cells andwells containing cells lysed with 0.1% Triton-X are used for baselinecontrols. Tamoxifen is used as a known control for each assay and DMSOonly will be run as vehicle control. The samples are incubated at ca.37° C. in humidified 5% CO₂ atmosphere for 72 hours. The plate ismonitored once a day during the incubation period, paying specialattention to the level of confluence. If the cells approach confluenceprior to the end of the 72 hour incubation period, the experiment isterminated and cell viability measured as described below.

Cell viability is determined using a commercially available kit todetermine ATP levels by luminescence. Briefly, the cell plate has themedia removed and replaced with 100 μL of fresh media, and the bufferand lyophilized substrate are equilibrated to room temperature. Thebuffer is used to reconstitute the substrate just prior to addition tothe wells of the cell plate (100 μL per well). The plate is placed intothe Infinite M200 plate reader, allowed to shake for 10 minutes followedby a 10 minute wait period. The plate is then read using an integrationtime of 0.5 sec with no attenuation.

The mean baseline controls (wells with Triton X-100 or no cells) aresubtracted from the total luminescence to give the net luminescence forthat well. This total is compared to the control of DMSO only. An IC₅₀is calculated as the concentration that led to a response of 50%compared to the vehicle control cells. Accordingly, those of ordinaryskill in the art can appreciate that the R configuration (at C-6) of theinstantly claimed composition are superior to other stereoisomers.

Table IV gives the IC₅₀ in various cell lines for compounds of theinvention.

TABLE IV IC50 IC50 IC50 Com- lung ovary Pancreas pound (A549) (Ovcar-3)(Capan-1) 18 139 uM (91%) 207 uM (89%) 192 uM (85%) 4 70 uM (97%) 100 uM(95%) 32 uM (93%) 3 84 uM (95%) 94 uM (90%) 168 uM (80%) 19 80 uM (98%)96 uM (98%) 32 uM (90%) 20 34 uM (99%) 60 uM (99%) 16 uM (99%) 21 17 uM(100%) 24 uM (100%) 23 uM (100%) 22 13 uM (100%) 20 uM (100%) 22 uM(100%) 10 170 uM (87%) 317 uM (52%) 277 uM (66%) 8 184 uM (80%) 221 uM(70%) 212 uM (75%) 23 1000 uM (7%) 1000 uM (35%) 775 uM (65%) 9 1000 uM(18%) 1000 uM (25%) 1000 uM (4%) 30 5 uM (100%) 29 uM (100%) 10 uM(100%) 31 7 uM (100%) 28 uM (100%) 15 uM (100%)

We claim:
 1. A compound of a formula:

wherein m is 4-8; n is 0-4; R₁-R₄ are hydrogen; Z is —O—; and the

symbol represents any type of bond regardless of stereochemistry.
 2. A compound according to claim 1 wherein both the C-13 methyl and C-17 hydroxyl are (S)-configured.
 3. A compound according to claim 1 wherein the C-6 substituent is (R)-configured.
 4. A pharmaceutical composition comprising a compound of claim 1 and a pharmaceutically-acceptable carrier. 